Formulations of hydroxypyridonate actinide/lanthanide decorporation agents

ABSTRACT

Provided herein are pharmaceutical formularions comprising a 1,2-HOPO chelating agent and/or 3,2-HOPO chelating agent.

STATEMENT OF GOVERNMENTAL SUPPORT

The invention was made with government support under National Instituteof Allergy and Infectious Diseases Contract #HHSN272201000046C andBiomedical Advanced Research and Development Authority Contract#IPIAA12OS99609, through the U.S. Department of Energy under Contract#DE-AC02-05CH11231. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage Application under 35 U.S.C.§ 371 of International Application No. PCT/US2017/050121, filed on Sep.5, 2017, designating the U.S. and published in English as WO 2018/048812A1 on Mar. 15, 2018, which claims the benefit of U.S. ProvisionalApplication No. 62/384,087, filed on Sep. 6, 2016, which is herebyincorporated by reference in its entirety.

BACKGROUND Field of the Invention

This invention relates generally to formulations for the treatment ofmetal poisoning.

Description of the Related Art

Exposure to radionuclides accidentally or deliberately scattered by aradiological dispersion device or deposited from a nuclear power plantaccident or nuclear device detonation could result in the contaminationof a large population. As internalized radionuclides are highly toxicand may cause both acute and chronic radiation injury, suchcontamination event would have dramatic public health consequences.Decorporation by chelating agents is the only way to reduce exposure ofcertain incorporated isotope, and diethylenetriaminepentaacetic acid(DTPA) has been the standard therapy for actinide/lanthanidedecorporation since its development and use by the U.S. Atomic EnergyCommission in the 1950's.

SUMMARY OF THE PREFERRED EMBODIMENTS

Embodiments herein provide for a pharmaceutical composition thatcomprises a 1,2-HOPO chelating agent in an amount from about 300 toabout 1500 mg; and sodium oleate. In some embodiments, the 1,2-HOPOchelating agent is 3,4,3-L1-1,2-HOPO. In some embodiments, sodium oleateis present at about 70 to about 130 mg. In some embodiments, sodiumoleate is present at 8 to 12% of a total weight of the composition. Insome embodiments, sodium oleate is about 11% of a total weight of thecomposition. In some embodiments, the 3,4,3-L1-1,2-HOPO chelating agentis present in an amount from 100 to 1500 mg. In some embodiments, the3,4,3-L1-1,2-HOPO chelating agent is present in an amount from 400 to1200 mg. In some embodiments, the 3,4,3-L1-1,2-HOPO chelating agent ispresent in an amount from 100 to 300 mg. In some embodiments, the3,4,3-L1-1,2-HOPO chelating agent is present in an amount of 600 mg. Insome embodiments, the 3,4,3-L1-1,2-HOPO chelating agent is present in anamount from 100 to 1500 mg. In some embodiments, the 3,4,3-L1-1,2-HOPOchelating agent is present in an amount from 400 to 1200 mg. In someembodiments, the 3,4,3-L1-1,2-HOPO chelating agent is present in anamount from 100 to 300 mg. In some embodiments, the 3,4,3-L1-1,2-HOPOchelating agent is present in an amount of 600 mg. In some embodiments,the pharmaceutical composition is packaged as a tablet. In someembodiments, the pharmaceutical composition is within a capsule. In someembodiments, the pharmaceutical composition is within one or moregranules. In some embodiments, the pharmaceutical composition ispackaged as a tablet. In some embodiments, the pharmaceuticalcomposition is within a capsule. In some embodiments, the pharmaceuticalcomposition is within one or more granules.

This is especially useful when administered to a subject that has beenexposed to, has been in contact with, or contaminated by one or moreknown or unknown actinides and/or lanthanides, or a mixture thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and others will be readily appreciated by theskilled artisan from the following description of illustrativeembodiments when read in conjunction with the accompanying drawings.

FIG. 1 shows the structures of 5-LIO(Me-3,2-HOPO) (“5LIO”) and3,4,3-L1(1,2-HOPO) (“343L1”).

FIG. 2 shows the structure of diethylenetriamine pentaacetic acid(DTPA).

FIG. 3 shows an embodiment of a chromatogram of API verification assaydiluent (9:1 water:ACN).

FIG. 4 shows an embodiment of a chromatogram of dissolution assaydiluent (SGF without enzyme, USP).

FIG. 5 shows an embodiment of a chromatogram of 3,4,3-L1(1,2-HOPO) 0.5mg/ml in API verification assay diluent.

FIG. 6 shows an embodiment of a chromatogram of 3,4,3-L1(1,2-HOPO) 1.1mg/ml in dissolution assay diluent.

FIG. 7 shows the appearance and packaging of embodiments of powder inbottle compositions A2 (left) and A11 (right).

FIG. 8 shows the appearance and packaging of embodiments of granulecompositions G11 (left) and G12 (right).

FIG. 9 shows the appearance and packaging of embodiments of chewabletablet compositions C11 (left), C13 (center) and C21 (right).

FIG. 10 shows the appearance and packaging of embodiments of tabletcompositions T50 (left) and T51 (right).

FIG. 11A shows the daily fecal excretion rate at necropsy of young adultfemale Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate.3,4,3-L1(1,2-HOPO) treatment was administered p.o. twice-daily oronce-daily for 6 days, starting at 24 hours post-exposure and mice wereeuthanized at 7 days.

FIG. 11B shows the daily urinary excretion rate at necropsy of youngadult female Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate.3,4,3-L1(1,2-HOPO) treatment was administered p.o. twice-daily oronce-daily for 6 days, starting at 24 hours post-exposure and mice wereeuthanized at 7 days.

FIG. 11C shows the cumulative excretion at necropsy on day 7 for FIG.11A and FIG. 11B.

FIG. 12A shows the daily fecal excretion rate at necropsy of young adultfemale Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate. Saline, DTPAor 3,4,3-L1(1,2-HOPO) treatment was administered i.p. or p.o. once-dailyfor 6 days, starting at 24 hours post-exposure and mice were euthanizedat 11 days.

FIG. 12B shows the daily urinary excretion rate at necropsy of youngadult female Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate.Saline, DTPA or 3,4,3-L1(1,2-HOPO) treatment was administered i.p. orp.o. once-daily for 6 days, starting at 24 hours post-exposure and micewere euthanized at 11 days.

FIG. 12C shows the cumulative excretion at necropsy on day 11 for FIG.12A and FIG. 12B.

FIG. 13A shows cumulative excretion in treated groups relative tocontrol at day 7 post-contamination for young adult female Swiss-Websterinjected i.v. with ²³⁸Pu-citrate. DTPA or 3,4,3-L1(1,2-HOPO) treatmentwas administered p.o. for six days, once-daily or twice-daily, startingat 24 hours post-exposure and mice were euthanized at 7 days.

FIG. 13B shows cumulative excretion in treated groups relative tocontrol at day 11 post-contamination for young adult femaleSwiss-Webster injected i.v. with ²³⁸Pu-citrate. DTPA or3,4,3-L1(1,2-HOPO) treatment was administered i.p. or p.o. for six days,once-daily, starting at 24 hours post-exposure and mice were euthanizedat 11 days.

FIG. 14A shows body, skeleton and liver retention in treated groups at 7days post-contamination for young adult female Swiss-Webster injectedi.v. with ²³⁸Pu-citrate. Saline, DTPA or 3,4,3-L1(1,2-HOPO) treatmentwas administered p.o. for six days, once-daily or twice-daily, startingat 24 hours post-exposure and mice were euthanized at 7 days.

FIG. 14B shows body, skeleton and liver retention in treated groups atday 11 post-contamination for young adult female Swiss-Webster injectedi.v. with ²³⁸Pu-citrate. Saline, DTPA or 3,4,3-L1(1,2-HOPO) treatmentwas administered i.p. or p.o. for six days, once-daily, starting at 24hours post-exposure and mice were euthanized at 11 days.

FIG. 15A shows the daily fecal excretion rate at necropsy of young adultmale Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate.3,4,3-L1(1,2-HOPO) treatment was administered p.o. twice-daily oronce-daily for 6 days, starting at 24 hours post-exposure and mice wereeuthanized at 7 days.

FIG. 15B shows the daily urinary excretion rate at necropsy of youngadult male Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate.3,4,3-L1(1,2-HOPO) treatment was administered p.o. twice-daily oronce-daily for 6 days, starting at 24 hours post-exposure and mice wereeuthanized at 7 days.

FIG. 15C shows the cumulative excretion at necropsy on day 7 for FIG.15A and FIG. 15B.

FIG. 16A shows the daily fecal excretion rate at necropsy of young adultmale Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate. Saline, DTPAor 3,4,3-L1(1,2-HOPO) treatment was administered i.p. or p.o. once-dailyfor 6 days, starting at 24 hours post-exposure and mice were euthanizedat 11 days.

FIG. 16B shows the daily urinary excretion rate at necropsy of youngadult male Swiss-Webster mice injected i.v. with ²³⁸Pu-citrate. Saline,DTPA or 3,4,3-L1(1,2-HOPO) treatment was administered i.p. or p.o.once-daily for 6 days, starting at 24 hours post-exposure and mice wereeuthanized at 11 days.

FIG. 16C shows the cumulative excretion at necropsy on day 11 for FIG.16A and FIG. 16B.

FIG. 17A shows cumulative excretion in treated groups relative tocontrol at day 7 post-contamination for young adult male Swiss-Websterinjected i.v. with ²³⁸Pu-citrate. DTPA or 3,4,3-L1(1,2-HOPO) treatmentwas administered p.o. for six days, once-daily or twice-daily, startingat 24 hours post-exposure and mice were euthanized at 7 days.

FIG. 17B shows cumulative excretion in treated groups relative tocontrol at day 11 post-contamination for young adult male Swiss-Websterinjected i.v. with ²³⁸Pu-citrate. DTPA or 3,4,3-L1(1,2-HOPO) treatmentwas administered i.p. or p.o. for six days, once-daily, starting at 24hours post-exposure and mice were euthanized at 11 days.

FIG. 18A shows body, skeleton and liver retention in treated groups at 7days post-contamination for young adult male Swiss-Webster injected i.v.with ²³⁸Pu-citrate. Saline, DTPA or 3,4,3-L1(1,2-HOPO) treatment wasadministered p.o. for six days, once-daily or twice-daily, starting at24 hours post-exposure and mice were euthanized at 7 days.

FIG. 18B shows body, skeleton and liver retention in treated groups atday 11 post-contamination for young adult male Swiss-Webster injectedi.v. with ²³⁸Pu-citrate. Saline, DTPA or 3,4,3-L1(1,2-HOPO) treatmentwas administered i.p. or p.o. for six days, once-daily, starting at 24hours post-exposure and mice were euthanized at 11 days.

FIG. 19A-FIG. 19F show data related to retention in liver and kidneysand excretion of radioactivity from [₁₄C]-3,4,3-L1(1,2-HOPO) in male andfemale mice after i.v., i.p., or p.o. administration. Data expressed asμg-eq (mean±SD, n=3) for tissue content and as percentage ofadministered dose (% AD) for excreta; excreta of each three-mouse groupwere pooled and standard deviations are not available.

FIG. 19A show data related to retention in liver and kidneys ofradioactivity from [₁₄C]-3,4,3-L1(1,2-HOPO) in male and female miceafter i.v. administration.

FIG. 19B show data related to excretion of radioactivity from[₁₄C]-3,4,3-L1(1,2-HOPO) in male and female mice after i.v.administration.

FIG. 19C show data related to retention in liver and kidneys ofradioactivity from [₁₄C]-3,4,3-L1(1,2-HOPO) in male and female miceafter i.p. administration.

FIG. 19D show data related to excretion of radioactivity from[₁₄C]-3,4,3-L1(1,2-HOPO) in male and female mice after i.p.administration.

FIG. 19E show data related to retention in liver and kidneys ofradioactivity from [₁₄C]-3,4,3-L1(1,2-HOPO) in male and female miceafter p.o. administration.

FIG. 19F show data related to excretion of radioactivity from[₁₄C]-3,4,3-L1(1,2-HOPO) in male and female mice after p.o.administration.

FIG. 20A-FIG. 20D show data related to retention and excretion ofradioactivity from [₁₄C]-3,4,3-L1(1,2-HOPO) in male and female ratsafter i.v. or p.o. administration. Groups of two or three rats wereadministered a single dose of [₁₄C]-3,4,3-L1(1,2-HOPO) and wereeuthanized at three time points between 2 hr and 24 hr postdose. Dataexpressed as μg-eq (mean±SD) for tissue content and as percentage ofadministered dose (% AD; mean±SD) for excreta.

FIG. 20A show data related to retention of radioactivity from[₁₄C]-3,4,3-L1(1,2-HOPO) in male and female rats after i.v.administration.

FIG. 20B show data related to excretion of radioactivity from[₁₄C]-3,4,3-L1(1,2-HOPO) in male and female rats after i.v.administration.

FIG. 20C show data related to retention of radioactivity from[₁₄C]-3,4,3-L1(1,2-HOPO) in male and female rats after p.o.administration.

FIG. 20D show data related to excretion of radioactivity from[₁₄C]-3,4,3-L1(1,2-HOPO) in male and female rats after p.o.administration.

FIG. 21A and FIG. 21B show time-course of mean plasma concentrations of3,4,3-L1(1,2-HOPO) in male (FIG. 21A) and female (FIG. 21B) beagle dogsafter a single oral dose of 37.5, 75, or 150 mg/kg 3,4,3-L1(1,2-HOPO).Mean (±SD) data (n≤3) are plotted against the nominal blood collectiontimes. Plasma concentrations from the 37.5 mg/kg group were not plottedat 6 hr because they were below the lower limit of quantification.

FIG. 21A shows clearance of 3,4,3-L1(1,2-HOPO) in plasma of male dogs.

FIG. 21B shows clearance of 3,4,3-L1(1,2-HOPO) in plasma of female dogs

FIG. 22 shows flux ratio comparison of 3,4,3-L1(1,2-HOPO) formulations.

FIG. 23 shows linearity results for 3,4,3-L1(1,2-HOPO) at T=0.

FIG. 24 shows linearity results for 3,4,3-L1(1,2-HOPO) at T=2 weeks.

FIG. 25 shows linearity results for 3,4,3-L1(1,2-HOPO) at T=4 weeks.

FIG. 26 shows linearity results for 3,4,3-L1(1,2-HOPO) at T=8 weeks.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

The potential consequences of a major radiological event are not onlylarge-scale external radiation exposure of the population, but alsouncontrolled dissemination of, and internal contamination with,radionuclides. When planning an emergency response to radiological andnuclear incidents, one must consider the need for treatment forcontaminated individuals. In addition to meeting the desired criteriafor post-exposure treatments such as safety, ease of administration, andbroad-spectrum efficacy against multiple radionuclides and levels ofchallenge, ideal countermeasures can include rapid onset; induce minimalto no performance-decrementing side effects; be compatible with currentmilitary Chemical, Biological, Radiological, Nuclear, and Explosivecountermeasures; and require minimal logistical burdens.Hydroxypyridinone-based actinide decorporation agents have shown themost promise as decorporation strategies for various radionuclides ofconcern, including the actinides plutonium and americium.

Provided herein are various formulations for decorporation agents.

The following disclosure provides a brief set of definitions, thenprovides further detail regarding the various formulations of thechelators provided herein, and then provides a set of Examples regardingvarious embodiments.

Definitions

The term “emergency” encompasses: (a) The event of an accidental releaseof the radioisotopes in the environment due to any nuclear accident. (b)Any accidental release of the hazardous nuclides in the environment. (c)A nuclear fallout including that occurring in the normal course of anexperimental, diagnostic or therapeutic purpose. (d) Any kind ofaccidental uptake and retention of the radionuclides by the human oranimal subjects. (e) Any other kind of exposure to the volatileradionuclides. (f) Any kind of a radiological accident.

The term “pharmaceutically acceptable salt,” as used herein, andparticularly when referring to a pharmaceutically acceptable salt of acompound, including 3,4,3-L1(1,2-HOPO), and refers to anypharmaceutically acceptable salts of a compound, and preferably refersto an acid addition salt of a compound.

The terms “pure,” “purified,” “substantially purified,” and “isolated”as used herein refer to the compound of the embodiment being free ofother, dissimilar compounds with which the compound, if found in itsnatural state, would be associated in its natural state. In certainembodiments described as “pure,” “purified,” “substantially purified,”or “isolated” herein, the compound can comprise at least 0.5% to 1%, 1%to 5%, 5% to 10%, 10% to 20%, 20% to 50%, 50% to 70%, 70% to 90%, 90% to95%, 95% to 99%, and 99% to 100%. In some embodiments, the amount of thecompound will be at least 50% or 75% of the mass, by weight, of a givensample. A “functional purity” is a measurement of the amount of aparticular compound in a sample or product in relation to othercompounds in a sample that can adversely impact the function of thecompound. Thus, other components in a sample that do not interfere withthe compound's activity (e.g., water), will not be used in determiningthe purity of a sample or product.

The terms “derivative,” “variant,” or other similar term refers to acompound that is an analog of the other compound.

The term “and/or” designates both the option of “and” as well as theoption of “or” in that particular circumstance. However, unlessotherwise specified in the specification, the use of the term “or” or“and” encompasses a description of both option as well. Thus, the use ofthe term “or” should not be taken as excluding the option of “and”,unless additional context indicates that it should (this definition doesnot apply to the language in the claims). The use of the singular orplural forms of a term encompasses both options (singlular or plural) aswell as both options combined (singular and plural), unless indicatedotherwise.

The term “inhibition” as used herein, refers to any statisticallysignificant decrease in the detrimental impact of the metal, includingfull blocking of the activity. For example, “inhibition” can refer to adecrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%in the detrimental impact of the metal.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

The terms “treat” or “prevent” do not require complete treatment orcomplete prevention under all conditions. A slowing of the onset of adisorder or its symptoms or a decrease in the number of the symptoms canbe adequate “prevention” in some embodiments. Similarly, a decrease inthe severity of the symptoms of the disorder can also be an effectivetreatment for a disorder. “Prophylactic treatment” denotes that thecompound is administered prior to exposure to the detrimental compound(e.g., metal such as plutonium or a MRI imaging agent). Treatment mayalso be in response to exposure, e.g., responsive therapy. Treat alsoencompasses remediation, decorporation, and/or decontamination.

“Therapeutically effective amount” means that amount of the chelatingagents, such as 3,4,3-L1(1,2-HOPO), 5-LIO(Me-3,2-HOPO) and/or DTPA, thatelicit the biological or medicinal response in a tissue system, animalor human sought by a researcher, veterinarian, medical doctor or otherclinician, which response includes alleviation of the symptoms of thedisease or disorder being treated. The specific amount of chelatingagents needed to elicit the biological or medicinal response will dependon a number of factors, including but not limited to the disease ordisorder being treated, the chelating agents being administered, themethod of administration, and the condition of the patient.

“Mammal” when used herein refers to any animal that is considered amammal. Preferably, the mammal is human.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), (incorporatedherein by reference).

The term “heavy metal” denotes one or more of a transition metal, ametalloid, a metallic element within groups 13, 14, and 15 of thePeriodic Table, an actinide or a lanthanide. Heavy metals include, forexample, gadolinium, lead, tin, cadmium, yttrium, scandium, andplutonium.

Pharmaceutical Formulations

In some embodiments, the pharmaceutical composition of formulationcomprises a chelating agent and one or more additional ingredient. Insome embodiments, the chelating agent is a 1,2-HOPO chelating agent. Insome embodiments, the chelating agent is 3,4,3-L1-1,2-HOPO.

In some embodiments, the pharmaceutical composition comprises a 1,2-HOPOchelating agent in an amount from about 300 to about 1500 mg and sodiumoleate. In some embodiments, the pharmaceutical composition the 1,2-HOPOchelating agent is 3,4,3-L1-1,2-HOPO.

In some embodiments, any amount of sodium oleate can be used, asappropriate for the intended use. In some embodiments, the amount ofsodium oleate present is between about 50 to about 150, for exampleabout 70 to about 130 mg. In some embodiments, sodium oleate is presentat about 5 to about 20% of a total weight of the composition, forexample, about 8 to 12% of a total weight of the composition or about11% of a total weight of the composition. Other amounts described hereinare also applicable for various applications.

In some embodiments, the 3,4,3-L1-1,2-HOPO chelating agent is present inan amount from about 50 to about 2000 mg, for example, about 100 to 1500mg, about 400 to 1200 mg, about 100 to 300 mg, or at about an amount of600 mg. Other amounts described herein are also applicable for variousapplications.

The amount of the chelating agents that may be combined with thepharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the subject treated and the particular mode ofadministration. Suitable dosage levels of the chelating agents includefrom about 1 mg to about 500 mg per kg body weight per day. In someembodiments, the suitable dosage level is from about 20 mg to about 100mg per kg body weight per day. In some embodiments, the suitable dosagelevel is from about 10 mol to about 100 μmol per kg body weight for3,4,3-L1-1,2-HOPO. In some embodiments, the suitable dosage level isfrom about 30 μmol to about 200 μmol per kg body weight for5-LIO-Me-3,2-HOPO. Dosage unit forms will generally contain from about20 mg to about 100 mg of the chelating agents. In addition, thepharmaceutical composition can be administered on an intermittent basis,i.e., at daily, semi-weekly, or weekly intervals. It will be understood,however, that the specific dose level for a particular subject willdepend on a variety of factors. These factors include the activity ofthe specific compound employed; the age, body weight, general health,sex, and diet of the subject; the time and route of administration andthe rate of excretion of the chelating agents; the combination ofchelating agents employed in the treatment; and, the severity of theparticular disease or condition for which therapy is sought.

In some embodiments, the pharmaceutical composition is packaged as atablet, within a capsule, and/or within one or more granules.

Suitable modes of administration of the pharmaceutical compositioninclude, but are not limited to, oral, topical, aerosol, inhalation byspray, parenteral, subcutaneous, intravenous, intramuscular,interperitoneal, rectal, and vaginal administration. The termparenteral, as used herein, includes subcutaneous injections, andintravenous, intrathecal, intramuscular, and intrasternal injection orinfusion techniques. A particular mode of administration is one thatbrings a compound of this invention to the actual or potential site(s)of radionuclide contamination in the subject. The pharmaceuticalcomposition can be in a solid, semi-solid, and/or liquid form. In someembodiments, any of the above formulations can be used for any of themetals provided herein.

In some embodiments, the formulation can include a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell known to those who are skilled in the art and are readilyavailable. In some embodiments, the carrier is chemically inert to acompound of this invention and has no detrimental side effects ortoxicity under the conditions of use. In some embodiments, thepharmaceutically acceptable carrier is free of pyrogen. Thepharmaceutically acceptable carriers which can be used include, but arenot limited to, water, glucose, lactose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, and urea.

The pharmaceutical compositions suitable for oral administrationinclude, but are not limited to, (a) liquid formulations; (b) capsules,sachets, tablets, lozenges, and troches, each containing a predeterminedamount of the active ingredient, as solids or granules; (c) powders; (d)suspensions; and (e) suitable emulsions. Liquid formulations may includediluents, such as water and alcohols, and optionally a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and the like. The tablet canfurther comprise one or more colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, or flavoringagents.

The pharmaceutical composition, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants (such asdichlorodifluoromethane, propane, nitrogen, and the like) ornon-pressured preparations (such as in a nebulizer or an atomizer). Whenthe site(s) of infection of a subject is the lungs, a preferred mode ofadministration is inhalation of an aerosol formulation either orally ornasally. in particular, the aerosol formulation may comprises particlesof a respirable size, including, but not limited to, mean particle sizesof 5 μm to 500 μm.

The pharmaceutical composition can be an injectable formulation. Therequirements for effective carriers for injectable compositions are wellknown to those of ordinary skill in the art (see, e.g., Pharmaceuticsand Pharmacy Practice, J. B. Lippincott Company, Philadelphia, Pa.,Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). In particularembodiments, injectable compositions are administered intravenously.Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The pharmaceutical composition can further comprise an excipient.Excipients that may be used include one or more carriers, surface activeagents, thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

In some embodiments, the pharmaceutical composition can comprise one ormore of the following formulations in table 1 and/or table 2:

TABLE 1 COMPOSITION OF EMBODIMENTS OF FORMULATIONS OF 3,4,3-LI(1,2-HOPO)Quantity in unit dosage form (mg) Prototype Immediate Capsules (500 mg)Capsules (100 mg) Powder for Release Chewable 3,4,3-LI(1,2-HOPO)3,4,3-LI(1,2-HOPO) Reconstitution Tablets Tablets Blend Lot#FLBN- BlendLot#FLBN- Formulation Formulation Formulation 20131029-1 - 0020131029-1 - 4 Ingredients ID# A11 (mg) ID# T51 (mg) ID# C21 (mg)gelatin capsule (mg) gelatin capsule (mg) 3,4,3-LI-(1,2- 500.0 500.0500.0 500.0 100.0 HOPO) Sodium Oleate 46.0 46.0 46.0  55.6  11.1Microcrystalline 500.0 — — — — Cellulose and Carboxymethyl Cellulose, NF(Avicel RC-591) Croscarmellose — 92.0 75.0 — — Sodium, NF (Ac-Di-Sol)Microcrystalline — 501.0 — — — Cellulose, NF (Avicel PH 102) Colloidalsilicone — 6.0 — — — dioxide, (Cab- O-Sil M5 P) Coprocessed — — 927.0 —— Microcrystalline Cellulose and Guar gum, (Avicel CE-15) Mannitol, — —927.0 — — (Mannogem Granular 2080) Magnesium — 6.0 25.0 — — stearate, NF(HyQual) Unit Weight (mg) 1046.0 1151.0 2500.0 555.6 111.1

TABLE 2 Dosage Form→ Powder Conventional Ingredients Formulation inBottle Granules Chewable Tablets Tablets ↓ ID→ A2 A11 G11 G12 C11 C13C21 T50 T51 Intra-Granular Materials 1.000 1.000 1.000 1.000 0.500 0.5000.500 0.500 0.500 (for granules and conventional tablets)3,4,3-Ll(1,2-HOPO) 0.092 0.092 0.092 0.092 0.046 0.046 0.046 0.046 0.046Sodium Oleate — 1.000 — — — — — — — Microcrystalline Cellulose — — 0.0750.075 0.075 — 0.075 0.084 0.092 and Carboxymethyl Cellulose, NF (AvivelRC-591) Croscarmellose Sodium, — — 1.833 — 1.854 — 0.927 — — NF(Ac-Di-Sol) Microcrystalline Cellulose — — — 1.533 — — — — — and guargum, NF (Avicel CE-15) Lactose Monohydrate, NF — — — — — 1.929 — — —(Pharmatose 300M) Lactose Monohydrate, — — — — — — 0.9227 — — Povidoneand Crospovidone, NF (Ludipress) Mannitol, USP (mannogem) — — — — 0.0250.025 0.025 — — Magnesium sterate, — — — — — — — 0.410 0.501 NF (HyQual)Microcrystalline Cellulose, — — — — — — — 0.005 0.006 NF (Avicel PH 102)Colloidal silicone dioxide, — — — — — — — — — NF (Cab-O-Sil M5P)Purified water, USP — — Q.S. Q.S. — — — — — Magensium sterate, NF — — —— — — — — — (HyQual) Extra-Granular Materials (for granules andconventional tablets) Hypromellose, 50 cps — — — 0.300 — — — — —Magensium sterate, NF — — — — — — — 0.005 0.006 (HyQual) Unit weight (g)1.092 2.0962 3.000 3.000 2.500 2.500 2.500 1.050 1.15

All of these oral dosage forms were found suitable for furtherdevelopment. Following extensive evaluation of dosage configuration,capsules containing 3,4,3-L1(1,2-HOPO) blended with excipient sodiumoleate were deemed to be the optimal dosage form for both the clinic anda potential mass casualty situation for the following reasons: 1) Thiscapsule dosage form allows more flexibility with regards to dose leveladjustments than tablet dosage forms. 2) Taste issues resulting inunwanted reactions are minimized in comparison to chewable tablets,powder in bottle dissolved in water, or dispersible granules dissolvedin water. 3) Capsules require less clean potable water and a greateraccuracy in dose administration than the other dosage forms. 4) Capsulesmay be adapted to pediatric formulations, with regards to dose leveladjustments and administration (by opening the capsules, and blendingthe powder with yoghurt or apple sauce-type mixtures.

In some embodiments, any of the ratios provided in any of theformulations can be maintained, while the amount of the activeingredient is increased or decreased.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more excipients.In some embodiments, the pharmaceutically suitable excipients include,but are not limited to, mannitol, lactose monohydrate, compressiblesugar, microcrystalline cellulose, hypromellose, povidone,pregelatinized starch, croscarmellose sodium, sodium starch glycolate,crospovidone, colloidal silicon dioxide, magnesium stearate,hydrogenated vegetable oil (type 1), and polysorbate 80.

In some embodiments, different forms of the formulation can be used,including i) powder, (ii) orally dispersible/dissolvable granules, (iii)chewable tablets, and/or (iv) conventional immediate release tablets.Based on the studies performed in the examples, nine formulationprototypes showed immediate drug release behavior and desired physicalproperties and were selected for API verification, gastric fluiddissolution, and related substance testing following defined liquidchromatography methods. Among these selected compositions, two arepowder in bottle formulations, two are granule formulations, three arechewable tablet formulations, and two are conventional tabletformulations. The respective compositions of these formulations aresummarized in the examples and in tables 1 and 2 above. A11 assaysconfirmed that these prototypes are suitable for further development.

In some embodiments, a powder formulation can comprise apharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO), sodiumoleate, and optionally, a mixture of microcrystalline cellulose andcarboxymethyl cellulose. In some embodiments, the composition caninclude 1 g of 3,4,3-L1(1,2-HOPO) and 0.092 g of sodium oleate. In someembodiments, the composition can include 1 g of 3,4,3-L1(1,2-HOPO),0.092 g of sodium oleate, and 1 g of microcrystalline cellulose andcarboxymethyl cellulose. In some embodiments, the composition caninclude 1 g of 3,4,3-L1(1,2-HOPO), 0.092 g of sodium oleate, and between0 g and 1 g of microcrystalline cellulose and carboxymethyl cellulose.Weights are listed in grams per unit of formulation.

In some embodiments, the formulation can be an orallydispersible/dissolvable granule formulation. It can include (by weightof ingredients per unit of formulation) 3,4,3-L1(1,2-HOPO)—1 g, Sodiumoleate—0.092 g, Croscarmellose Sodium—0.075 g, and microcrystallinecellulose & guar gum—1.833 g. In some embodiments, the formulation caninclude 3,4,3-L1(1,2-HOPO)—1 g, Sodium oleate—0.092 g, croscarmelloseSodium—0.075 g, lactose monohydrate—1.533 mg/ml, and hypromellose—0.3 g.

In some embodiments, an orally dispersible/dissolvable granuleformulation can comprise a pharmaceutically suitable composition of3,4,3-L1(1,2-HOPO), sodium oleate, croscarmellose sodium, a mixture ofmicrocrystalline cellulose and guar gum, lactose monohydrate, andhypromellose. In some embodiments, the composition can include 1 g of3,4,3-L1(1,2-HOPO), 0.092 g of sodium oleate, 0.075 g of croscarmellosesodium, between 0 and 1.833 g of microcrystalline cellulose and guargum, between 0 and 1.533 mg/ml of lactose monohydrate, and between 0 and0.3 g of hypromellose. A11 weights are listed in grams per unit offormulation, unless otherwise noted.

In some embodiments, the formulation can be a chewable tabletformulation. In some embodiments, a chewable tablet formulation cancomprise a pharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO),sodium oleate, croscarmellose sodium, a mixture of microcrystallinecellulose and guar gum, magnesium stearate, a mixture of lactosemonohydrate, povidone, and crospovidone, and mannitol. In someembodiments, the composition can include 0.5 g of 3,4,3-L1(1,2-HOPO),0.046 g of sodium oleate, 0.075 g of croscarmellose sodium, 1.854 g ofmicrocrystalline cellulose and guar gum, and 0.025 g of magnesiumstearate. In some embodiments, the composition can include 0.5 g of3,4,3-L1(1,2-HOPO), 0.046 g of sodium oleate, 1.929 g of lactosemonohydrate, povidone, and crospovidone, and 0.025 g of magnesiumstearate. In some embodiments, the composition can include 0.5 g of3,4,3-L1(1,2-HOPO), 0.046 g of sodium oleate, 0.075 g of croscarmellosesodium, 0.927 g of microcrystalline cellulose and guar gum, 0.9227 gmannitol, and 0.025 g of magnesium stearate. In some embodiments, thecomposition can include 0.5 g of 3,4,3-L1(1,2-HOPO), 0.046 g of sodiumoleate, between 0 and 0.075 g of croscarmellose sodium, between 0 and1.854 g of microcrystalline cellulose and guar gum, 0.025 g of magnesiumstearate, between 0 and 1.929 g of lactose monohydrate, povidone, andcrospovidone, and between 0 and 0.9227 g of mannitol. A11 weights arelisted in grams per unit of formulation.

In some embodiments, an immediate release tablet formulation cancomprise a pharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO),sodium oleate, croscarmellose sodium, microcrystalline cellulose,colloidal silicon dioxide, and magnesium stearate. In some embodiments,the composition can include 0.5 g of 3,4,3-L1(1,2-HOPO), 0.046 g ofsodium oleate, 0.084 g of croscarmellose sodium, 0.41 g ofmicrocrystalline cellulose, 0.005 g of colloidal silicon dioxide, and0.005 g of magnesium stearate. In some embodiments, the composition caninclude 0.5 g of 3,4,3-L1(1,2-HOPO), 0.046 g of sodium oleate, 0.092 gof croscarmellose sodium, 0.501 g of microcrystalline cellulose, 0.006 gof colloidal silicon dioxide, and 0.006 g of magnesium stearate. In someembodiments, the composition can include 0.5 g of 3,4,3-L1(1,2-HOPO),0.046 g of sodium oleate, between 0.084 and 0.092 g of croscarmellosesodium, between 0.41 g and 0.501 g of microcrystalline cellulose,between 0.005 g and 0.006 g of colloidal silicon dioxide, and between0.005 g and 0.006 g of magnesium stearate. A11 weights are listed ingrams per unit of formulation.

In some embodiments, the formulations can be stable under 25° C./60% RHand 40° C./75% RH storage conditions over six months or longer. Suchstable formulations can be a powder formulation, a chewable tabletformulation, an immediate release tablet formulation, a 500 mg capsuleformulation, and a 100 mg capsule formulation. The respectivecompositions of these prototype formulations are summarized below.

In some embodiments, a powder formulation can comprise apharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO), sodiumoleate, and a mixture of microcrystalline cellulose and carboxymethylcellulose. In some embodiments, the composition can include 500 mg of3,4,3-L1(1,2-HOPO), 46 mg of sodium oleate, and 500 mg ofmicrocrystalline cellulose and carboxymethyl cellulose. A11 weights arelisted in milligrams per unit of formulation.

In some embodiments, a chewable tablet formulation can comprise apharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO), sodiumoleate, croscarmellose sodium, a mixture of microcrystalline celluloseand guar gum, mannitol, and magnesium stearate. In some embodiments, thecomposition can include 500 mg of 3,4,3-L1(1,2-HOPO), 46 mg of sodiumoleate, 75 mg of croscarmellose sodium, 927 mg of microcrystallinecellulose and guar gum, 927 mg of mannitol, and 25 mg of magnesiumstearate. A11 weights are listed in milligrams per unit of formulation.

In some embodiments, an immediate release tablet formulation cancomprise a pharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO),sodium oleate, croscarmellose sodium, microcrystalline cellulose,colloidal silicon dioxide, and magnesium stearate. In some embodiments,the composition can include 500 mg of 3,4,3-L1(1,2-HOPO), 46 mg ofsodium oleate, 92 mg of croscarmellose sodium, 501 mg ofmicrocrystalline cellulose, 6 mg of colloidal silicon dioxide, and 6 mgof magnesium stearate. Weights are listed in milligrams per unit offormulation.

In some embodiments, a 500 mg capsule formulation can be provided thatincludes a pharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO)and sodium oleate. In some embodiments, the composition can include 500mg of 3,4,3-L1(1,2-HOPO) and 55.6 mg of sodium oleate.

In some embodiments, a 100 mg capsule formulation can be provided thatcomprises a pharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO)and sodium oleate. In some embodiments, the composition can include 100mg of 3,4,3-L1(1,2-HOPO) and 11.1 mg of sodium oleate.

The formulations can be configured for administration parenterally (ip)or orally (po) via consecutive injections or gavages at various doses,including, but not limited to: human doses of 2.5, 12.5, 25, and 50μmol/kg, using the accepted conversion system of mouse doses into humanequivalent doses—HED—based on body surface area). The parenteralformulation can be pure 3,4,3-L1(1,2-HOPO), while the oral formulationcan include a 90:10 weight ratio of 3,4,3-L1(1,2-HOPO) and sodiumoleate.

In some embodiments, an intraperitoneal injection formulation cancomprise a pharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO).In some embodiments, the composition can include 3,4,3-L1(1,2-HOPO) at aconcentration of 30 μmol/kg ip (equivalent approximate human dose: 2.5μmol/kg).

In some embodiments, an oral formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and sodium oleate. In someembodiments, the composition can include a 90:10 weight ratio of3,4,3-L1(1,2-HOPO) and sodium oleate. In some embodiments, thecomposition can include 3,4,3-L1(1,2-HOPO) at a concentration of 150μmol/kg po. In some embodiments, the composition can include3,4,3-L1(1,2-HOPO) at a concentration of 300 μmol/kg po. In someembodiments, the composition can include 3,4,3-L1(1,2-HOPO) at aconcentration of 300 μmol/kg po. In some embodiments, the compositioncan include 3,4,3-L1(1,2-HOPO) at a concentration between 150 μmol/kg poand 600 μmol/kg po.

In some embodiments, an intraperitoneal injection, oral, or intravenousinjection formulation can comprise a pharmaceutically suitablecomposition of [₁₄C]-3,4,3-L1(1,2-HOPO) and sodium oleate. In someembodiments, the composition can include [₁₄C]-3,4,3-L1(1,2-HOPO) at adosage of 1 μmol/kg and 0% sodium oleate. In some embodiments, thecomposition can include [₁₄C]-3,4,3-L1(1,2-HOPO) at a dosage of 1μmol/kg and 10% sodium oleate. In some embodiments, the composition caninclude [₁₄C]-3,4,3-L1(1,2-HOPO) at a dosage of 1800 μmol/kg and 0%sodium oleate. In some embodiments, the composition can include[₁₄C]-3,4,3-L1(1,2-HOPO) at a dosage of 1800 μmol/kg and 10% sodiumoleate. In some embodiments, the composition can include[₁₄C]-3,4,3-L1(1,2-HOPO) at a dosage between 1 μmol/kg and 1800 μmol/kgand between 0 and 10% sodium oleate.

In some embodiments, an orally administrated capsule formulation cancomprise a pharmaceutically suitable composition of 3,4,3-L1(1,2-HOPO).In some embodiments, the composition can include 50 μmol/kg of3,4,3-L1(1,2-HOPO). In some embodiments, the composition can include 100μmol/kg of 3,4,3-L1(1,2-HOPO). In some embodiments, the composition caninclude 200 μmol/kg of 3,4,3-L1(1,2-HOPO). In some embodiments, thecomposition can include between 50 μmol/kg and 200 μmol/kg of3,4,3-L1(1,2-HOPO).

As outlined in the examples (Examples 10-11), the enhancement of thepermeability of the active pharmaceutical ingredient 3,4,3-L1(1,2-HOPO)using oral permeation enhancers was evaluated. Fifteen differentpermeation enhancers were evaluated for their ability at increasing thepermeability of 3,4,3-L1(1,2-HOPO), using an in vitro PAMPA assay withartificial GIT lipid membranes. A significant increase was observed inpermeability for one formulation containing 10 mg/mL of Polysorbate 80and 1 mg/mL of API. All other tested formulations showed no or minorimprovement in permeability.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more excipient. Insome embodiments, the excipients may include, but are not limited to,sodium lauryl sulfate, caprolactam, polysorbate 80, sodium deoxycholate,isopropyl myristate, 1-phenylpiperazine, piperine, menthone, labrafaclipophile WL 1349, gelucire 44/14, labrafil M2130 CS, labrafil M2125 CS,maisine 35-1, peceol, labrasol, sodium decyl sulfate, sodium octylsulfate, decyltrimethylammonium bromide, span-80 (Sorbitan monooleate),Triton X-100, sodium glycocholate hydrate, cholic acid, heptanoic acid,isopropyl palmitate, methyl laurate, sodium oleate, urea,1-octyl-2-pyrrolidone, 1-methylpiperazine, 1-methyl-2-pyrrolidinone,n-caproic acid, sodium salicylate, (±)-limonene, L-fenchone, cineole,pinene oxide, 2-octyl-1-dodecanol, cumin seed oil, caproyl PGMC, caproyl90 (propylene glycol dicaprylate), lauroglycol FCC, lauroglycol 90,labrafac PG, transcutol, gelucire 50/13, and labrafil M1944 CS.

In some embodiments, the composition can include 3,4,3-L1(1,2-HOPO) at aconcentration of 1 mg/ml and polysorbate 80 at a concentration of 10mg/ml. In some embodiments, the composition can include3,4,3-L1(1,2-HOPO) at a concentration of 1 mg/ml and 2-octyl-1-dodecanolat a concentration of 2.5 mg/ml. In some embodiments, the compositioncan include 3,4,3-L1(1,2-HOPO) at a concentration of 1 mg/ml and sodiumoleate at a concentration of 2.5 mg/ml.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more excipients.In some embodiments, pharmaceutically suitable excipients include, butare not limited to, sodium oleate, sodium lauryl sulfate, caprolactam,polysorbate 80, sodium deoxycholate, isopropyl myristate,1-phenylpiperazine, piperine, menthone, labrafac lipophile, gelucire44/14, labrafil M2130 CS, labrafil M2125 CS, maisine 35-1, peceol,labrasol, sodium decyl sulfate, sodium octyl sulfate,decyltrimethylammonium bromide, span-80 (sorbitan monooleate), tritonX-100, sodium glycocholate hydrate, cholic acid, heptanoic acid,isopropyl palmitate, methyl laurate, sodium oleate, urea,1-octyl-2-pyrrolidone, 1-methylpiperazine, 1-methyl-2-pyrrolidinone,n-caproic acid, sodium salicylate, (±)-limonene, L-fenchone, cineole,pinene oxide, 2-octyl-1-dodecanol, cumin seed oil, caproyl PGMC, caproyl90 (Propylene glycol dicaprylate), lauroglycol FCC, lauroglycol 90,labrafac PG, transcutol, gelucire 50/13, labrafil M1944 CS, mannitol,compressible sugar, coprocessed microcrystalline cellulose and guar gum,coprocessed lactose monohydrate and povidone, microcrystallinecellulose, lactose monohydrate, povidone, HPMC, hypromellose,pregelatinized starch, croscarmellose sodium, sodium starch glycolate,crospovidone, colloidal silicon dioxide, magnesium stearate,microcrystalline cellulose and carboxymethyl cellulose, hydrogenatedvegetable oil, type 1, co-processed lactose monohydrate, povidone andcrospovidone, co-processed microcrystalline cellulose and carboxymethylcellulose, maltodextrin, sodium citrate, and/or sodium chloride.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more permeationenhancers. In some embodiments, the permeation enhancers include, butare not limited to, sodium oleate, sodium lauryl sulfate, caprolactam,polysorbate 80, sodium deoxycholate, isopropyl myristate,1-phenylpiperazine, piperine, menthone, labrafac lipophile, gelucire44/14, labrafil M2130 CS, labrafil M2125 CS, maisine 35-1, peceol,labrasol, sodium decyl sulfate, sodium octyl sulfate,decyltrimethylammonium bromide, span-80 (sorbitan monooleate), tritonX-100, sodium glycocholate hydrate, cholic acid, heptanoic acid,isopropyl palmitate, methyl laurate, sodium oleate, urea,1-octyl-2-pyrrolidone, 1-methylpiperazine, 1-methyl-2-pyrrolidinone,n-caproic acid, sodium salicylate, (±)-limonene, L-fenchone, cineole,pinene oxide, 2-octyl-1-dodecanol, cumin seed oil, caproyl PGMC, caproyl90 (Propylene glycol dicaprylate), lauroglycol FCC, lauroglycol 90,labrafac PG, transcutol, gelucire 50/13, and/or labrafil M1944 CS.

In some embodiments, the composition can include between 1% and 10%sodium oleate by weight. In some embodiments, the composition caninclude between 5 mg and 100 mg of sodium oleate.

In some embodiments, the composition can include sodium lauryl sulfateat a concentration of 0.1 mg/ml. In some embodiments, the compositioncan include caprolactam at a concentration of 2.5 mg/ml.

In some embodiments, the composition can include polysorbate 80 at aconcentration of 2.5 mg/ml. In some embodiments, the composition caninclude polysorbate 80 at a concentration of 2 mg/ml. In someembodiments, the composition can include polysorbate 80 at aconcentration of 10 mg/ml. In some embodiments, the composition caninclude polysorbate 80 at a concentration between 2 mg/ml and 10 mg/ml.

In some embodiments, the composition can include sodium deoxycholate ata concentration of 2.5 mg/ml. In some embodiments, the composition caninclude sodium deoxycholate at a concentration of 10 mg/ml. In someembodiments, the composition can include sodium deoxycholate at aconcentration between 2.5 mg/ml and 10 mg/ml.

In some embodiments, the composition can include isopropyl myristate ata concentration of 2.5 mg/ml. In some embodiments, the composition caninclude isopropyl myristate at a concentration of 10 mg/ml. In someembodiments, the composition can include isopropyl myristate at aconcentration between 2.5 mg/ml and 10 mg/ml.

In some embodiments, the composition can include 1-phenylpiperazine at aconcentration of 2.5 mg/ml.

In some embodiments, the composition can include piperine at aconcentration of 2.5 mg/ml.

In some embodiments, the composition can include menthone at aconcentration of 2.5 mg/ml.

In some embodiments, the composition can include labrafac lipophile WL1349 at a concentration of 5 mg/ml.

In some embodiments, the composition can include gelucire 44/14 at aconcentration of 5 mg/ml. In some embodiments, the composition caninclude gelucire 44/14 at a concentration of 20 mg/ml. In someembodiments, the composition can include gelucire 44/14 at aconcentration between 1 mg/ml and 40 mg/ml.

In some embodiments, the composition can include labrafil M2130 CS at aconcentration of 5 mg/ml. In some embodiments, the composition caninclude labrafil M2125 CS at a concentration of 5 mg/ml.

In some embodiments, the composition can include maisine 35-1 at aconcentration of 5 mg/ml. In some embodiments, the composition caninclude maisine 35-1 at a concentration of 20 mg/ml. In someembodiments, the composition can include maisine 35-1 at a concentrationbetween 1 mg/m and 40 mg/ml.

In some embodiments, the composition can include peceol 35-1 at aconcentration of 5 mg/ml. In some embodiments, the composition caninclude peceol 35-1 at a concentration of 20 mg/ml. In some embodiments,the composition can include peceol 35-1 at a concentration between 1mg/m and 40 mg/ml.

In some embodiments, the composition can include labrasol at aconcentration of 5 mg/ml. In some embodiments, the composition caninclude sodium decyl sulfate at a concentration of 0.2 mg/ml. In someembodiments, the composition can include sodium octyl sulfate at aconcentration of 0.2 mg/ml. In some embodiments, the composition caninclude decyltrimethylammonium bromide at a concentration of 1 mg/ml. Insome embodiments, the composition can include span-80 (sorbitanmonooleate) at a concentration of 2.5 mg/ml. In some embodiments, thecomposition can include triton X-100 at a concentration of 2.5 mg/ml. Insome embodiments, the composition can include sodium glycocholatehydrate at a concentration of 1.0 mg/ml. In some embodiments, thecomposition can include cholic acid at a concentration of 2.5 mg/ml. Insome embodiments, the composition can include heptanoic acid at aconcentration of 2.5 mg/ml.

In some embodiments, the composition can include isopropyl palmitate ata concentration of 2.5 mg/ml. In some embodiments, the composition caninclude methyl laurate at a concentration of 2.5 mg/ml. In someembodiments, the composition can include sodium oleate at aconcentration of 2.5 mg/ml. In some embodiments, the composition caninclude urea at a concentration of 2.5 mg/ml. In some embodiments, thecomposition can include 1-octyl-2-pyrrolidone at a concentration of 2.5mg/ml. In some embodiments, the composition can include1-methylpiperazine at a concentration of 2.5 mg/ml. In some embodiments,the composition can include 1-methyl-2-pyrrolidinone at a concentrationof 2.5 mg/ml. In some embodiments, the composition can include n-caproicacid at a concentration of 2.5 mg/ml. In some embodiments, thecomposition can include sodium salicylate at a concentration of 2.5mg/ml. In some embodiments, the composition can include (±)-limonene ata concentration of 2.5 mg/ml. In some embodiments, the composition caninclude L-fenchone at a concentration of 2.5 mg/ml. In some embodiments,the composition can include cineole at a concentration of 2.5 mg/ml. Insome embodiments, the composition can include pinene oxide at aconcentration of 2.5 mg/ml. In some embodiments, the composition caninclude 2-octyl-1-dodecanol at a concentration of 2.5 mg/ml. In someembodiments, the composition can include cumin seed oil at aconcentration of 2.5 mg/ml. In some embodiments, the composition caninclude caproyl PGMC at a concentration of 5 mg/ml. In some embodiments,the composition can include caproyl 90 (propylene glycol dicaprylate) ata concentration of 5 mg/ml.

In some embodiments, the composition can include lauroglycol FCC at aconcentration of 5 mg/ml. In some embodiments, the composition caninclude lauroglycol 90 at a concentration of 5 mg/ml. In someembodiments, the composition can include labrafac PG at a concentrationof 5 mg/ml. In some embodiments, the composition can include transcutolat a concentration of 5 mg/ml. In some embodiments, the composition caninclude gelucire 50/13 at a concentration of 5 mg/ml. In someembodiments, the composition can include labrafil M1944 CS at aconcentration of 5 mg/ml.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more diluents. Insome embodiments, the diluents include, but are not limited to,mannitol, compressible sugar, coprocessed microcrystalline cellulose andguar gum, coprocessed lactose monohydrate and povidone, microcrystallinecellulose, and lactose monohydrate. In some embodiments, the compositioncan include 10% diluent by weight. In some embodiments, the compositioncan include 70% diluent by weight. In some embodiments, the compositioncan include between 10% and 70% diluent by weight. In some embodiments,the composition can include mannitol at a concentration of 2 mg/ml. Insome embodiments, the composition can include compressible sugar at aconcentration of 2 mg/ml.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more binders. Insome embodiments, the binders include, but are not limited to, povidone,HPMC, hypromellose, and pregelatinized starch. In some embodiments, thecomposition can include 10% binder by weight. In some embodiments, thecomposition can include 70% binder by weight. In some embodiments, thecomposition can include between 10% and 70% binder by weight.

In some embodiments, the composition can include povidone at aconcentration of 2 mg/ml. In some embodiments, the composition caninclude hypromellose at a concentration of 2 mg/ml. In some embodiments,the composition can include pregelatinized starch at a concentration of2 mg/ml.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or moredisintegrants. In some embodiments, the disintegrants include, but arenot limited to, croscarmellose sodium, sodium starch glycolate, andcrospovidone. In some embodiments, the composition can include 2%disintegrant by weight. In some embodiments, the composition can include8% disintegrant by weight. In some embodiments, the composition caninclude between 2% and 8% disintegrant by weight.

In some embodiments, the composition can include croscarmellose sodiumat a concentration of 2 mg/ml. In some embodiments, the composition caninclude sodium starch glycolate at a concentration of 2 mg/ml. In someembodiments, the composition can include crospovidone at a concentrationof 2 mg/ml.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more lubricantsand glidants. In some embodiments, the lubricants and glidants include,but are not limited to, colloidal silicon dioxide and magnesiumstearate. In some embodiments, the composition can include 0.2%lubricant and glidant by weight. In some embodiments, the compositioncan include 20% lubricant and glidant by weight. In some embodiments,the composition can include between 0.2% and 20% lubricant and glidantby weight.

In some embodiments, the composition can include colloidal silicondioxide at a concentration of 2 mg/ml. In some embodiments, thecomposition can include magnesium stearate at a concentration of 2mg/ml.

In some embodiments, a formulation can comprise a pharmaceuticallysuitable composition of 3,4,3-L1(1,2-HOPO) and one or more otherexcipients. In some embodiments, the other excipients include, but arenot limited to, microcrystalline cellulose and carboxymethyl cellulose,microcrystalline cellulose and guar gum, hydrogenated vegetable oil,type 1, co-processed lactose monohydrate, povidone and crospovidone,co-processed microcrystalline cellulose and carboxymethyl cellulose,maltodextrin, sodium citrate, and sodium chloride.

In some embodiments, the composition can include hydrogenated vegetableoil type 1 at a concentration of 2 mg/ml. In some embodiments, thecomposition can include sodium citrate at a concentration of 0.008 M. Insome embodiments, the composition can include sodium chloride at aconcentration of 0.14 M.

The 1,2-HOPO and 3,2-HOPO chelating agents suitable for use in thepresent invention are taught in U.S. Pat. No. 4,698,431(“Hydroxypyridonate Chelating Agents”), U.S. Pat. No. 5,634,901(“3-Hydroxy-2(1H)-pyridonate Chelating Agents”), and U.S. Pat. No.5,892,029 (“3-Hydroxy-2(1H)-pyridonate Chelating Agents”), all of whichare hereby incorporated by reference.

Suitable 1,2-HOPO chelating agent include, but are not limited to,molecules defined by the structure:

wherein R is a hydroxy group or where R₁ and R₂ are selected from thegroup consisting of H, —CH₃, —CH₂CH₃ and —CH₂φ, and X is eitherhydrogen, an alkali metal ion, or a quaternary ammonium ion.

Suitable 1,2-HOPO chelating agent include, but are not limited to,molecules incorporating a plurality of HOPO-type structures, including:

wherein 1, m and n are integers between one and twenty. In a particularembodiment of the invention, m is three. In a particular embodiment ofthe invention, m is three and n is four. In a particular embodiment ofthe invention, 1 and n are three, and m is four.

Suitable 1,2-HOPO and 3,2-HOPO chelating agents include, but are notlimited to, a chelating agent comprised of a plurality of chelatingfunctional units joined by one or more linking members, said chelatingfunctional units independently selected from the group consisting of

in which at least one of said plurality of chelating functional units onsaid chelating agent is

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁₋₄ aliphatic hydrocarbon groups, and C₁₋₄ aliphatichydrocarbon groups substituted by a single halide, hydroxy, carboxy,acrylamido group or an aryl group, and R′ is a member selected from thegroup consisting of a bond to a linking member, a hydrogen atom, C₁₋₈aliphatic hydrocarbon groups, aryl groups, and C₁₋₈ aliphatichydrocarbon groups substituted by amino, carboxy, or hydroxy groups.

Suitable 3,2-HOPO chelating agents include, but are not limited to, achelating agent having the structure:

wherein R₁ is a member selected from the group consisting of hydrogen,C₁₋₄ aliphatic hydrocarbon groups, and C₁₋₄ aliphatic hydrocarbon groupssubstituted by a single halide, hydroxy, carboxy, or aryl group; Z is amember selected from the group consisting of O, NH, N-alkyl, and N-aryl;a is 2-4; and b is 2-4.

A suitable 1,2-HOPO and a suitable 3,2-HOPO are shown in FIG. 1 .

The methods for synthesizing the 1,2-HOPO and 3,2-HOPO chelating agentsare taught in U.S. Pat. Nos. 4,698,431; 5,634,901; and 5,892,029, all ofwhich are hereby incorporated by reference.

The chelating agents are capable of binding or chelating, or capable offorming stable complexes with actinides and/or lanthanides, such as thecations of Eu, Pu, Np, Th, Am, and/or Cf, such as of ¹⁵²Eu(III),²⁴¹Am(III), ²³⁸Pu(IV), ²³⁷Np(IV), ²³⁷Np(V), and ²³³U(VI).

Embodiments provided herein include prodrugs of the chealtors. Suchprodrugs are in general functional derivatives of the compounds that arereadily convertible in vivo into the required compound. Thus, in themethods, the term “administering” shall encompass the treatment of thevarious disorders described with the compound specifically disclosed orwith a compound which may not be specifically disclosed, but whichconverts to the specified compound in vivo after administration to asubject in need thereof. Conventional procedures for the selection andpreparation of suitable prodrug derivatives are described, for example,in Wermuth, “Designing Prodrugs and Bioprecursors,” in Wermuth, ed., ThePractice of Medicinal Chemistry, 2nd Ed., pp. 561-586 (Academic Press2003). Prodrugs include esters that hydrolyze in vivo (for example inthe human body) to produce a compound of this invention or a saltthereof. Suitable ester groups include, without limitation, thosederived from pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moiety preferably has no more than sixcarbon atoms. Illustrative esters include formates, acetates,propionates, butyrates, acrylates, citrates, succinates, andethylsuccinates.

Methods of Use

In some embodiments, a method for treating a subject for a heavy metalexposure is provided. The method comprises administering atherapeutically effective amount of a pharmaceutical formulationcomprising a 1,2-HOPO chelating agent to a subject that has an excessamount of one or more of heavy metals, actinides and/or lanthanides, ora mixture thereof. Additional options for therapies are also provided inU.S. Pat. Pub. No. 20120214843, the entirety of which is herebyincorporated by reference. Methods of treatment can include treating asubject in need by administering a therapeutically effective amount ofone or more pharmaceutical compositions comprising a chelating agent (asprovided herein) to a subject in need of such treatment. In someembodiments the subject has been exposed to, have been in contact with,or contaminated by one or more known or unknown actinides and/orlanthanides, or a mixture thereof.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. A11 publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “achelating agent” includes a plurality of such chelating agents, and soforth.

The invention having been described, the following examples are offeredto illustrate the subject invention by way of illustration, not by wayof limitation.

EXAMPLES Example 1-3,4,3-L1(1,2-HOPO)-Excipient Compatibility StudySummary

The objective of the analytical study described in this report was toevaluate the interactions between 3,4,3-L1(1,2-HOPO) and selectedpharmaceutical excipients under 25° C./60% RH and 40° C./75% RHconditions over eight weeks. Physical appearance and potency of thedifferent samples were assessed at T=0, 2, 4, and 8 weeks through visualobservations and high performance liquid chromatography (HPLC) analysis.

The HPLC system suitability and linearity were verified at each timepoint and were within the protocol requirements. Among the 14 compoundstested, 4 excipients (pregelatinized starch, compressible sugar,providone, and hydrogenated vegetable oil) resulted in a decrease in3,4,3-L1(1,2-HOPO) purity or in an increase of specific impuritycontent. These results will be taken into consideration for furtherinvestigation on the bioavailability enhancement of 3,4,3-L1(1,2-HOPO).

1. Purpose of Study

The purpose of this study was to provide data that can be used tosupport research efforts. It was not conducted in accordance with U.S.Food and Drug Administration (FDA) “Good Laboratory Practice forNonclinical Laboratory Studies” (GLP) regulations, as described in 21CFR Part 58. However, the study was planned, performed, recorded, andreported in accordance with standard practices to ensure data qualityand integrity.

2. Objective of Study

The objective of this study was to evaluate the interactions between3,4,3-L1(1,2-HOPO) and selected pharmaceutical excipients under 25°C./60% RH and 40° C./75% RH conditions over eight weeks, including:

a. Mannitol

b. Lactose Monohydrate

c. Compressible sugar

d. Microcrystalline Cellulose

e. Hypromellose

f. Povidone

g. Pregelatinized Starch

h. Croscarmellose Sodium

i. Sodium Starch Glycolate

j. Crospovidone

k. Colloidal silicon dioxide

l. Magnesium stearate

m. Hydrogenated Vegetable Oil, Type 1

n. Polysorbate 80 (PS), NF (Spectrum Chemicals, Cat #PO138)

3. Experimental Design

The stability of 3,4,3-L1(1,2-HOPO) was tested according to theconditions listed below in TABLE 1.1. A11 test samples were storedthroughout the study in 40 mL clear USP Type 1 glass vials (28 mm OD×95mm height; 24 mm screw cap), wrapped in aluminum foil.

TABLE 1.1 CONDITIONS FOR TESTING STABILITY OF STABILITY OF3,4,3-LI(1,2-HOPO) Test Time Points Condition T = 0 2 weeks 4 weeks 8weeks Extra Samples 40° C./75% RH X X X X 2 vials 25° C./60% RH X X X 2vials X test represents: 1. Visual Observation (color and physical form)2. Assay and Chromatographic Purity

TABLE 1.2 shows the list of excipients, and various drug-excipientratios that were used in the study.

TABLE 1.2 LIST OF EXCIPIENTS AND VARIOUS DRUG- EXCIPIENT RATIOS USED INTHE STUDY Quantity to API- be Filled in No. of Sample Excipient eachVial (mg) Vials to No. Excipient Name* Category Ratio** API Excipient befilled 1 Mannitol Diluent 1:1 50 50 11 (MA) 2 Lactose monohydrateDiluent 1:1 50 50 11 (LM) 3 Compressible sugar Diluent 1:1 50 50 11 (CS)4 Microcrystalline Cellulose Diluent 1:1 50 50 11 (MCC) 5 HypromelloseBinder 1:1 50 50 11 (HYP) 6 Povidone Binder 1:1 50 50 11 (PVP) 7Pregelatinized Starch Binder 1:1 50 50 11 (PGS) 8 Croscarmellose sodiumDisintegrant 1:1 50 50 11 (CCS) 9 Sodium Starch Glycolate Disintegrant1:1 50 50 11 (SSG) 10 Crospovidone Disintegrant 1:1 50 50 11 (CPVP) 11Colloidal silicon dioxide Lubricant/   1:0.2 50 10 11 (CSD) Glidant 12Magnesium Stearate Lubricant/   1:0.2 50 10 11 MGS) Glidant 13Hydrogenated Vegetable Oil Lubricant/   1:0.2 50 10 11 (HVO) Glidant 14Polysorbate 80 Permeation   1:0.2 50 10 11 (PS) Enhancer 15 Control:3,4,3-LI(1,2-HOPO) API 1:0 50 0 11 (API) *These excipients are generallyused in the development of chewable tablets, orally dispersible tablets(ODT), and mouth dissolving tablets. **For placebo preparations of eachcombination, the specified amount of excipient was weighed separatelyinto one vial per condition, to the exception of the API control.4. Materials And Methods

a. Test and Control Articles

Test Article: 3,4,3-L1(1,2-HOPO)

Manufacturer: Ash Stevens, Inc. (Detroit, Mich.)

Lot Number: ML-11-276

Physical Description: Pale yellow solid

Storage Conditions: Refrigerated 2-8° C. protected from light.

Materials:

Purified Water HPLC Grade - Supplier: Ricca Hydrochloric acid ACSGrade - Supplier: EMD Sodium hydroxide ACS Grade - Supplier: BDH FormicAcid HPLC Grade - Supplier: EMD Acetonitrile HPLC Grade - Supplier:Fischer HPLC Column Agilent, Eclipse XDB-C18, 4.6 × 150 mm, 5 μmHypromellose, Substitution Type Spectrum Chemicals, Cat# HY122 2910, 50mPa · s, USP Mannitol, USP Spectrum Chemicals, Cat# MA165 LactoseMonohydrate, Powder, NF Spectrum Chemicals, Cat# LA106 MicrocrystallineCellulose, NF Spectrum Chemicals, Cat# C1679 Compressible sugar, NFDomino Specialty Ingredients Povidone K-29/32, USP Plasdone K29/32, ISPTechnologies Pregelatinized Starch, NF (Starch Colorcon, Inc 1500)Croscarmellose sodium, NF Spectrum Chemicals, Cat# C1366 Sodium StarchGlycolate, Type-A, Spectrum Chemicals, Cat# S1962 pH 5.5 to 7.5, NFCrospovidone, NF Polyplasdone, ISP Technologies Colloidal silicondioxide, NF Spectrum Chemicals, Cat# S1510 Magnesium stearate, NFSpectrum Chemicals, Cat# MA130 Hydrogenated Vegetable Oil, Type 1,Lubritab, JRS Pharma NF Polysorbate 80, NF Spectrum Chemicals, Cat#PO138

-   Test Samples: Each test mixture was prepared by weighing the desired    quantity of 3,4,3-L1(1,2-HOPO) and excipient (both screened through    sieve #40) into vials. Both ingredients were first mixed using a    clean glass rod and then vortexed. At each time interval, one vial    was withdrawn from each drug-excipient series along with the    corresponding placebo preparation and tested as described below.

b. Sample Characterization

-   Visual Observation: For each sample solution, visual observation    consisted in recording color and physical form.

c. Chromatographic Assay and Purity Assessment

-   Standard Stock Solutions: For each standard stock solutions, the    test article was weighed (200 mg) and dissolved by sonication into    30 mL of diluent (water:acetonitrile=90%:10%). After equilibration    at room temperature, the volume of the standard solution was    adjusted to 50 mL. Standard stock solutions were prepared in    duplicates, working standard solutions were prepared by dilution of    each stock with the diluent to the desired concentrations.-   Calibration Standards: With each experiment, 5 calibration standard    solutions at different concentrations were prepared from stock    solutions using the diluent. Concentrations of the calibration    standards were between 1.6 and 2.4 mg/mL. The calibration standard    solutions were chromatographed to demonstrate the linearity of the    calibration curve over the concentration range.-   Chromatographic Purity: For test article purity evaluation, one of    the calibration standard solutions prepared above was used.-   Sample Preparation: For each sample, 25 mL of diluent was added to    the sample vial to reach a final concentration of 2 mg/mL of    3,4,3-L1(1,2-HOPO). The vials were then mechanically shaken for 15    minutes, followed by centrifugation of the sample solutions (10,000    rpm, 10 min), and the supernatants were used for the assay.-   Placebo Preparation: 25 mL of diluent was added to each of the    placebo vial. The vials were then mechanically shaken for 15    minutes, followed by centrifugation of the sample solutions (10,000    rpm, 10 min), and the supernatants were used for the assay.    Analytical Method:    Instrument: Waters Alliance 2695 liquid chromatography system    Column: Agilent, Eclipse XDB-C18, 4.6×150 mm, 5 μm.    Mobile Phase A: 0.05% formic acid in 95% H2O: 5% ACN    Mobile Phase B: 0.05% formic acid in acetonitrile (ACN)

TABLE 1.3 GRADIENT CONDITIONS Time (min) A % B % 0.00 100 0 30.00 60 4040.00 0 100 41.00 100 0 50.10 100 0Column Temperature: 25° C.Flow Rate: 1.0 mL/min.Injection Volume: 10 μLDetection: 250 nmRun Time: 50 minDiluent: 9:1 H₂O:ACNAnalysis Sequence:

TABLE 1.4 ANALYSIS SEQUENCE Sample Name #of Injections Diluent 1Standard Solution (System Suitability) 6 Diluent 1 Linearity Solution -1 through 5 1 (for each concentration) Diluent 1 Samples (Not more thantwelve (12) Each sample 1 injection injections) Bracketing Standard(Standard Solution) 1 Excipient Placebos 1 (for each placebo) At the endof the samples, inject bracketing 1

-   Suitability Requirements: There should be no interference from the    diluent/blank at the retention times of 3,4,3-L1(1,2-HOPO) peaks.    The relative standard deviation (% RSD) for five replicate system    suitability injections should be below 2.0%. The response factor of    the second standard should be within 95-105%. The correlation    coefficient (R₂) of the system linearity standards should be higher    than 0.990.    5. Results

a. System Suitability

System suitability and linearity results are summarized in TABLE 1.5 toTABLE 1.8 and FIG. 23 to FIG. 26 for all time points (T=0 (FIG. 23 ), 2(FIG. 24 ), 4 (FIG. 25 ), and 8 (FIG. 26 ) weeks). A11 systemsuitability and linearity results were within the protocol requirements.The prepared calibration standard curves were found to be linear, andthe correlation coefficients are included in the tables, together withcalibration curves.

TABLE 1.5 SYSTEM SUITABILITY FOR 3,4,3-LI(1,2-HOPO) AT T = 0 StandardWeight Std. ml Dilution % Details taken (mg) Dilution (ml) Taken Vol(ml) Purity 200.77 50 5 10 100 Bracketing S.No. std Area Recovery 18631074 100.5 2 8633151 100.5 3 8428130 98.2 Details 343LI Peak AreaStandard-1 8535609 Standard-2 8541521 Standard-3 8596940 Standard-48652568 Standard-5 8592901 Standard-6 8600027 Average 8586594 SD 43153 %RSD 0.50 Linearity Concentration Peak Samples (mg/mL) Area Linearity1.61 6730552 Standard 1 Linearity 1.81 7645145 Standard 2 Linearity 2.018534785 Standard 3 Linearity 2.21 9344601 Standard 4 Linearity 2.4110267523 Standard 5 Slope 4369875 Intercept −268877 R squared 1.000

TABLE 1.6 SYSTEM SUITABILITY FOR 3,4,3-LI(1,2-HOPO) AT T = 2 WEEKSStandard Weight Std. ml Dilution % Details taken (mg) Dilution (ml)Taken Vol (ml) Purity 202.86 50 5 10 100 Bracketing S.No. std AreaRecovery 1 8643151 101.1 2 8678578 101.5 3 8670629 101.4 4 8538994 99.9Details 3LIO Peak Area Standard-1 8425238 Standard-2 8547444 Standard-38562554 Standard-4 8579837 Standard-5 8570076 Standard-6 8605815 Average8548494 SD 63442 % RSD 0.74 Linearity Concentration Peak Samples (mg/mL)Area Linearity 1.62 6791128 Standard 1 Linearity 1.83 7701103 Standard 2Linearity 2.03 8624832 Standard 3 Linearity 2.23 9455860 Standard 4Linearity 2.43 10369590 Standard 5 Slope 4393020 Intercept −323178 Rsquared 1.000

TABLE 1.7 SYSTEM SUITABILITY FOR 3,4,3-LI(1,2-HOPO) AT T = 4 WEEKSStandard Weight Std. ml Dilution % Details taken (mg) Dilution (ml)Taken Vol (ml) Purity 200.09 50 5 10 100 Bracketing S.No. std AreaRecovery 1 8546199 100.6 2 8567449 100.9 3 8531088 100.4 4 8526160 100.43LIO Details Peak Area Standard-1 8375801 Standard-2 8504122 Standard-38521310 Standard-4 8494189 Standard-5 8518087 Standard-6 8548076 Average8493598 SD 60536 % RSD 0.71 Linearity Concentration Peak Samples (mg/mL)Area Linearity 1.60 6756483 Standard 1 Linearity 1.80 7708440 Standard 2Linearity 2.00 8594007 Standard 3 Linearity 2.20 9472736 Standard 4Linearity 2.40 10304210 Standard 5 Slope 4427882 Intercept −292575 Rsquared 0.999

TABLE 1.8 SYSTEM SUITABILITY FOR 3,4,3-LI(1,2-HOPO) AT T = 8 WEEKSStandard Weight Std. ml Dilution % Details taken (mg) Dilution (ml)Taken Vol (ml) Purity 200.34 50 5 10 100 Bracketing S.No. std AreaRecovery 1 8523708 100.7 2 8507536 100.6 3 8521355 100.7 4 8376145 99.0Details 3LIO Peak Area Standard-1 8344005 Standard-2 8430555 Standard-38506911 Standard-4 8531787 Standard-5 8408894 Standard-6 8540557 Average8460452 SD 78455 % RSD 0.93 Linearity Concentration Peak Samples (mg/mL)Area Linearity 1.60 6688971 Standard 1 Linearity 1.80 770585 Standard 2Linearity 2.00 8553209 Standard 3 Linearity 2.20 9394501 Standard 4Linearity 2.40 10326009 Standard 5 Slope 4474090 Intercept −429817 Rsquared 0.999

b. Stability Determination

Results of the compatibility study are summarized in TABLE 1.9 to TABLE1.23. The test article 3,4,3-L1(1,2-HOPO) was stable over 8 weeks underthe described conditions (25° C./60% RH and 40° C./75% RH) in thecontrol samples. Most excipient-API mixtures displayed similarstability, to the exception of the mixtures including pregelatinizedstarch (TABLE 1.15) and hydrogenated vegetable oil (TABLE 1.21) thatresulted in apparent decrease of 3,4,3-L1(1,2-HOPO) HPLC purity. Inaddition an increase in specific purities was observed for theexcipient-API mixtures containing pregelatinized starch (TABLE 1.15),compressible sugar (TABLE 1.11), providone (TABLE 1.14), andhydrogenated vegetable oil (TABLE 1.21).

TABLE 1.9 3,4,3-LI(1,2-HOPO)-MANNITOL COMPATIBILITY Initial 40° C./75%RH 25° C./60% RH Test (T = 0) 2 Weeks 4 weeks 8 Weeks 2 Weeks 4 Weeks 8Weeks Visual Pale yellow Pale yellow Pale yellow Pale yellow Pale yellowPale yellow Pale yellow Observation powder pasty pasty pasty powderpowder powder material material material Identification RT matches RTmatches RT matches RT matches RT matches RT matches RT matches by HPLCwith with with with with with with (3LIO) standard standard standardstandard standard standard standard Related Substances (% Area. n = I)3LIO Purity 98.77 98.73 98.67 98.50 98.77 98.80 98.57 Total 1.23 1.271.33 1.50 1.23 1.20 1.43 Impurities Unknown Impurities RRT % w/w RRT %w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown 0.24 —0.24 0.03 0.24 0.04 0.24 0.05 0.25 0.03 0.24 0.03 0.24 0.03 Unknown 0.580.03 0.58 0.06 0.58 0.08 0.58 0.08 0.58 0.02 0.58 0.04 0.58 0.03 Unknown0.59 0.04 0.59 0.05 0.6 0.06 0.59 0.06 0.59 0.03 0.6 0.04 0.59 0.03Unknown 0.62 0.03 0.62 0.05 0.62 0.06 0.62 0.06 0.62 0.03 0.62 0.04 0.620.04 Unknown 0.81 — 0.81 0.02 0.81 0.02 0.81 0.02 — — — — — — Unknown0.87 0.09 0.87 0.14 0.87 0.15 0.87 0.16 0.87 0.09 0.87 0.12 0.87 0.1Unknown 0.89 0.04 0.89 0.03 0.89 0.02 0.89 0.02 0.89 0.05 0.89 0.03 0.890.03 Unknown 1.03 0.2 1.03 0.06 1.02 0.04 1.03 0.09 1.03 0.05 1.02 0.041.03 0.07 Unknown 1.11 0.04 1.11 0.05 1.11 0.05 1.11 0.06 1.11 0.05 1.110.05 1.11 0.05 Unknown 1.27 0.3 1.27 0.28 1.27 0.23 1.27 0.36 1.27 0.361.27 0.24 1.27 0.5 Unknown 1.3 0.23 1.3 0.24 1.3 0.27 1.3 0.21 1.3 0.211.3 0.26 1.3 0.18 Unknown 1.39 0.06 1.39 0.06 1.39 0.05 1.39 0.04 1.40.05 1.39 0.05 1.39 0.06 Unknown 1.41 0.03 1.41 0.02 1.41 0.01 1.41 0.011.41 0.06 1.41 0.02 1.41 0.02 Unknown 1.46 0.11 1.46 0.08 1.46 0.07 1.460.07 1.47 0.08 1.46 0.06 1.46 0.08 Unknown 1.72 — — — 1.72 0.07 1.730.08 — — 1.72 0.07 1.73 0.03 Unknown 2.31 0.04 2.31 0.04 2.3 0.04 2.310.06 2.32 0.05 2.3 0.05 2.31 0.09 Unknown 2.6 — 2.6 0.04 2.59 0.03 2.60.04 2.61 0.04 2.59 0.04 2.6 0.06 Unknown 2.8 — 2.8 0.03 2.79 0.03 2.80.03 2.82 0.03 2.79 0.03 2.8 0.04

TABLE 1.10 3,4,3-LI(1,2-HOPO)-LACTOSE MONOHYDRATE COMPATIBILITY 40°C./75% RH 25° C./60% RH Test Initial (T = 0) 2 Weeks 4 weeks 8 Weeks 2Weeks 4 Weeks 8 Weeks Visual Pale yellow Pale yellow Pale yellow Paleyellow Pale yellow Pale yellow Pale yellow Observation powder pastymaterial pasty material pasty material powder powder powderIdentification by RT matches RT matches RT matches RT matches RT matchesRT matches RT matches HPLC (3LIO) with standard with standard withstandard with standard with standard with standard with standard RelatedSubstances (% Area. n = I) 3LIO Purity 98.79 98.64 98.65 98.45 98.7498.72 98.45 Total Impurities 1.21 1.36 1.35 1.55 1.26 1.28 1.55 RRT %w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w UnknownImpurities Unknown 0.24 — 0.24 0.13 0.24 0.04 0.24 0.05 0.25 0.03 0.240.03 0.24 0.03 Unknown 0.58 0.05 0.58 0.09 0.58 0.07 0.58 0.08 0.57 0.050.58 0.06 0.58 0.05 Unknown 0.59 0.05 0.59 0.07 0.6 0.06 0.59 0.06 0.590.05 0.6 0.05 0.59 0.05 Unknown 0.62 0.04 0.62 0.08 0.62 0.06 0.62 0.070.62 0.04 0.62 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.03 0.81 0.02 0.810.02 — — — — — — Unknown 0.87 0.12 0.87 0.12 0.87 0.15 0.87 0.15 0.870.13 0.87 0.14 0.87 0.13 Unknown 0.89 0.03 0.89 0.13 0.89 0.02 0.89 0.020.89 0.04 0.89 0.02 0.89 0.02 Unknown 1.03 0.19 1.02 0.07 1.03 0.04 1.030.08 1.03 0.08 1.02 0.04 1.03 0.07 Unknown 1.11 0.04 1.11 0.05 1.11 0.061.11 0.05 1.11 0.05 1.11 0.05 1.11 0.05 Unknown 1.27 0.23 1.27 0.34 1.270.26 1.27 0.41 1.27 0.31 1.27 0.27 1.27 0.52 Unknown 1.3 0.23 1.3 0.081.3 0.27 1.3 0.2 1.3 0.22 1.3 0.26 1.3 0.19 Unknown 1.39 0.05 1.39 0.031.39 0.05 1.39 0.06 1.4 0.04 1.39 0.05 1.39 0.05 Unknown 1.41 0.01 1.410.01 1.41 0.01 1.41 0.01 1.41 0.02 1.41 0.01 1.41 0.01 Unknown 1.46 0.131.46 0.06 1.46 0.07 1.46 0.08 1.47 0.06 1.46 0.07 1.46 0.1 Unknown 1.72— — — 1.72 0.07 1.73 0.07 — — 1.72 0.06 1.73 0.03 Unknown 2.31 0.03 2.310.03 2.3 0.04 2.31 0.07 2.32 0.05 2.3 0.04 2.31 0.08 Unknown 2.6 — 2.60.03 2.59 0.03 2.6 0.04 2.61 0.04 2.59 0.03 2.6 0.05 Unknown 2.8 — 2.80.03 2.79 0.02 2.8 0.03 2.82 0.03 2.79 0.03 2.8 0.04

TABLE 1.11 3,4,3-LI(1,2-HOPO)-COMPRESSIBLE SUGAR COMPATIBILITY 40°C./75% RH 25° C./60% RH Test Initial (T = 0) 2 Weeks 4 weeks 8 Weeks 2Weeks 4 Weeks 8 Weeks Visual Pale yellow Brown pasty Brown pasty Brownpasty Pale yellow Pale yellow Pale yellow Observation powder materialmaterial material powder powder powder Identification by RT matches RTmatches RT matches RT matches RT matches RT matches RT matches HPLC(3LIO) with standard with standard with standard with standard withstandard with standard with standard Related Substances (% Area. n = I)3LIO Purity 98.77 98.24 98.27 98.42 98.69 98.73 98.49 Total Impurities1.23 1.76 1.73 1.58 1.31 1.27 1.51 RRT % w/w RRT % w/w RRT % w/w RRT %w/w RRT % w/w RRT % w/w RRT % w/w Unknown Impurities Unknown 0.24 — 0.240.09 0.24 0.01 0.24 0.06 0.25 0.03 0.24 0.03 0.24 0.03 Unknown 0.45 —0.45 0.41 0.45 0.37 — — — — — — — — Unknown 0.58 0.06 0.57 0.09 0.58 0.10.58 0.08 0.57 0.05 0.58 0.06 0.58 0.06 Unknown 0.59 0.05 0.59 0.08 0.60.08 0.59 0.07 0.59 0.05 0.6 0.05 0.59 0.05 Unknown 0.62 0.05 0.62 0.070.62 0.08 0.62 0.07 0.62 0.04 0.62 0.05 0.62 0.05 Unknown 0.81 — 0.810.02 0.81 0.03 0.81 0.02 — — — — — — Unknown 0.87 0.11 0.87 0.14 0.870.13 0.87 0.15 0.87 0.12 0.87 0.14 0.87 0.14 Unknown 0.89 0.03 0.89 0.070.89 0.1 0.89 0.02 0.89 0.04 0.89 0.02 0.89 0.02 Unknown 1.03 0.19 1.030.08 1.02 0.04 1.03 0.18 1.03 0.06 1.02 0.03 1.03 0.09 Unknown 1.11 0.041.11 0.05 1.11 0.06 1.11 0.06 1.11 0.05 1.11 0.05 1.11 0.05 Unknown 1.270.24 1.27 0.32 1.27 0.28 1.27 0.38 1.27 0.37 1.27 0.27 1.27 0.44 Unknown1.3 0.23 1.3 0.12 1.3 0.08 1.3 0.18 1.3 0.22 1.3 0.25 1.3 0.22 Unknown1.39 0.06 1.39 0.03 1.39 0.03 1.39 0.04 1.4 0.04 1.39 0.05 1.39 0.05Unknown 1.4 0.01 1.41 0.01 1.41 0.01 1.41 0.01 1.41 0.02 1.41 0.01 1.410.01 Unknown 1.46 0.14 1.46 0.11 1.46 0.08 1.46 0.06 1.47 0.09 1.46 0.071.46 0.09 Unknown 1.72 — — — 1.72 0.09 1.73 0.07 — — 1.72 0.05 1.73 0.04Unknown 2.31 0.04 2.31 0.04 2.3 0.03 2.31 0.07 2.32 0.04 2.3 0.04 2.310.08 Unknown 2.6 — 2.6 0.03 2.59 0.03 2.6 0.04 2.61 0.05 2.59 0.04 2.60.05 Unknown 2.8 — 2.8 0.03 2.79 0.02 2.8 0.03 2.82 0.03 2.79 0.03 2.80.04

TABLE 1.12 3,4,3-LI(1,2-HOPO)-MICROCRYSTALLINE CELLULOSE COMPATIBILITY40° C./75% RH 25° C./60% RH Test Initial (T = 0) 2 Weeks 4 weeks 8 Weeks2 Weeks 4 Weeks 8 Weeks Visual Pale yellow Pale yellow Pale yellow Paleyellow Pale yellow Pale yellow Pale yellow Observation powder pastymaterial pasty material pasty material powder powder powderIdentification by RT matches RT matches RT matches RT matches RT matchesRT matches RT matches HPLC (3LIO) with standard with standard withstandard with standard with standard with standard with standard RelatedSubstances (% Area. n = I) 3LIO Purity 98.79 98.43 98.53 98.42 98.6498.73 98.48 Total Impurities 1.21 1.57 1.47 1.58 1.36 1.27 1.52 RRT %w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w UnknownImpurities Unknown 0.24 — 0.24 0.17 0.24 0.04 0.24 0.05 0.25 0.04 0.240.04 0.24 0.03 Unknown 0.58 0.06 0.58 0.1 0.58 0.08 0.58 0.08 0.57 0.050.58 0.07 0.58 0.06 Unknown 0.59 0.05 0.59 0.08 0.6 0.05 0.58 0.06 0.590.05 0.6 0.05 0.59 0.05 Unknown 0.62 0.05 0.62 0.09 0.62 0.06 0.62 0.060.62 0.04 0.60 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.05 0.81 0.02 0.810.02 — — — — — — Unknown 0.87 0.12 0.87 0.12 0.87 0.15 0.87 0.14 0.870.11 0.87 0.15 0.87 0.14 Unknown 0.89 0.03 0.89 0.27 0.89 0.03 0.89 0.020.89 0.04 0.89 0.02 0.89 0.02 Unknown 1.03 0.18 1.03 0.05 1.03 0.03 1.030.1 1.03 0.07 1.03 0.06 1.03 0.08 Unknown 1.11 0.04 1.13 0.07 1.11 0.061.11 0.06 1.11 0.05 1.11 0.05 1.11 0.05 Unknown 1.27 0.21 1.27 0.27 1.270.32 1.27 0.44 1.27 0.43 1.27 0.24 1.27 0.5 Unknown 1.3 0.25 1.3 0.091.3 0.25 1.3 0.18 1.3 0.18 1.3 0.26 1.3 0.21 Unknown 1.39 0.05 1.39 0.031.39 0.06 1.39 0.05 1.39 0.06 1.39 0.04 1.39 0.04 Unknown 1.41 0.01 1.410.01 1.41 0.02 1.41 0.01 1.41 0.03 1.41 0.01 1.41 0.01 Unknown 1.46 0.121.46 0.07 1.46 0.11 1.46 0.09 1.47 0.09 1.47 0.06 1.46 0.08 Unknown 1.72— — — 1.72 0.07 1.73 0.06 — — 1.72 0.07 1.73 0.03 Unknown 2.31 0.04 2.310.03 2.3 0.05 2.31 0.07 2.32 0.06 2.3 0.04 2.31 0.08 Unknown 2.6 — 2.60.03 2.59 0.05 2.6 0.04 2.61 0.05 2.59 0.04 2.6 0.05 Unknown 2.8 — 2.80.03 2.79 0.03 2.8 0.03 2.81 0.03 2.79 0.03 2.8 0.04

TABLE 1.13 3,4,3-LI(1,2-HOPO)-HYPROMELLOSE COMPATIBILITY 40° C./75% RH25° C./60% RH Test Initial (T = O) 2 Weeks 4 weeks 8 Weeks 2 Weeks 4weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellow Pale PalePale Observation yellow pasty pasty pasty yellow yellow yellow powdermaterial material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3LIO) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3LIO Purity 96.75 98.39 98.53 98.39 98.69 98.69 98.34Total Impurities 1.25 1.61 1.47 1.61 1.31 1.31 1.66 RRT % w/w RRT % w/wRRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.16 0.24 0.04 0.24 0.06 0.24 0.03 0.24 0.03 0.240.03 Unknown 0.58 0.05 0.58 0.1 0.58 0.08 0.58 0.09 0.57 0.05 0.58 0.070.58 0.06 Unknown 0.59 0.05 0.59 0.08 0.6 0.06 0.59 0.07 0.59 0.05 0.60.05 0.59 0.05 Unknown 0.62 0.04 0.62 0.08 0.62 0.06 0.62 0.08 0.62 0.040.62 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.05 0.81 0.02 0.81 0.02 — — — —— — Unknown 0.87 0.09 0.87 0.09 0.87 0.15 0.87 0.15 0.87 0.12 0.87 0.140.87 0.13 Unknown 0.89 0.03 0.89 0.23 0.89 0.03 0.89 0.03 0.89 0.03 0.890.02 0.89 0.02 Unknown 1.03 0.19 1.03 0.05 1.02 0.03 1.03 0.09 1.03 0.081.02 0.04 1.03 0.08 Unknown 1.11 0.04 1.13 0.08 1.11 0.05 1.11 0.07 1.110.05 1.11 0.05 1.11 0.05 Unknown 1.27 0.29 1.27 0.37 1.27 0.32 1.27 0.431.27 0.36 1.27 0.27 1.27 0.59 Unknown 1.3 0.2 1.3 0.1 1.3 0.24 1.3 0.111.3 0.22 1.3 0.26 1.3 0.19 Unknown 1.39 0.06 1.39 0.05 1.39 0.08 1.390.09 1.39 0.05 1.39 0.05 1.39 0.05 Unknown 1.41 0.01 1.41 0.01 1.41 0.011.4 0.01 1.41 0.02 1.41 0.01 1.41 0.01 Unknown 1.46 0.18 1.46 0.09 1.460.12 1.46 0.12 1.47 0.1 1.46 0.09 1.46 0.15 Unknown 1.72 — — — 1.72 0.071.73 0.08 — — 1.72 0.06 1.73 0.03 Unknown 2.31 0.03 2.31 0.03 2.3 0.032.31 0.06 2.32 0.05 2.3 0.03 2.31 0.07 Unknown 2.6 — 2.6 0.03 2.59 0.042.6 0.04 2.61 0.04 2.59 0.04 2.6 0.06 Unknown 2.8 — 2.8 0.03 2.79 0.032.8 0.03 2.81 0.03 2.79 0.03 2.8 0.04

TABLE 1.14 3,4,3-LI(1,2-HOPO)-POVIDONE COMPATIBILITY 40° C./75% RH 25°C./60% RH Test Initial (T = O) 2 Weeks 4 weeks 8 Weeks 2 Weeks 4 weeks 8Weeks Visual Pale Pale yellow Pale yellow Pale yellow Pale Pale PaleObservation yellow pasty pasty pasty yellow yellow yellow powdermaterial material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3LIO) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3LIO Purity 98.77 98.63 98.60 98.27 98.62 98.60 98.40Total Impurities 1.23 1.37 1.40 1.73 1.38 1.40 1.60 RRT % w/w RRT % w/wRRT %w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.08 0.24 0.04 0.24 0.05 0.24 0.03 0.24 0.04 0.240.03 Unknown 0.58 0.05 0.58 0.08 0.58 0.07 0.58 0.08 0.57 0.04 0.58 0.070.58 0.06 Unknown 0.59 0.05 0.59 0.07 0.6 0.05 0.59 0.06 0.59 0.05 0.60.05 0.59 0.05 Unknown 0.62 0.04 0.62 0.07 0.62 0.06 0.62 0.07 0.62 0.040.62 0.06 0.62 0.05 Unknown 0.81 — 0.81 0.03 0.81 0.02 0.81 0.02 — — — —— — Unknown 0.87 0.1 0.87 0.11 0.87 0.14 0.87 0.15 0.87 0.12 0.87 0.150.87 0.14 Unknown 0.89 0.03 0.89 0.1 0.89 0.03 0.89 0.02 0.89 0.04 0.890.03 0.89 0.02 Unknown 1.03 0.18 1.02 0.07 1.03 0.03 1.03 0.11 1.02 0.081.03 0.04 1.03 0.07 Unknown 1.11 0.03 1.11 0.06 1.11 0.05 1.11 0.06 1.110.05 1.11 0.05 1.11 0.05 Unknown 1.27 0.27 1.27 0.37 1.27 0.3 1.27 0.511.27 0.43 1.27 0.27 1.27 0.54 Unknown 1.3 0.23 1.3 0.09 1.3 0.25 1.30.17 1.3 0.18 1.3 0.29 1.3 0.19 Unknown 1.39 0.06 1.39 0.04 1.39 0.081.39 0.1 1.39 0.08 1.39 0.06 1.39 0.07 Unknown 1.41 0.01 1.41 0.01 1.410.01 1.4 0.01 1.41 0.03 1.41 0.01 1.41 0.01 Unknown 1.46 0.14 1.46 0.091.46 0.1 1.46 0.14 1.47 0.1 1.46 0.1 1.46 0.11 Unknown 1.72 — — — 1.720.05 1.73 0.05 — — 1.72 0.09 1.73 0.03 Unknown 2.31 0.03 2.31 0.04 2.30.03 2.31 0.06 2.32 0.04 2.3 0.04 2.31 0.07 Unknown 2.6 — 2.6 0.03 2.590.04 2.6 0.05 2.61 0.05 2.59 0.03 2.6 0.06 Unknown 2.8 — 2.8 0.03 2.790.03 2.8 0.03 2.81 0.03 2.79 0.03 2.8 0.04

TABLE 1.15 3,4,3-LI(1,2-HOPO)-PREGELATINIZED STARCH COMPATIBILITY 40°C./75% RH 25° C./60% RH Test Initial (T = O) 2 Weeks 4 Weeks 8 Weeks 2Weeks 4 Weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellowPale Pale Pale Observation yellow pasty pasty pasty yellow yellow yellowpowder material material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3LIO) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3LIO Purity 98.78 98.55 98.55 97.56 98.66 98.69 98.32Total Impurities 1.22 1.45 1.45 2.44 1.34 1.31 1.68 RRT % w/w RRT % w/wRRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.11 0.24 0.05 0.24 0.36 0.24 0.03 0.24 0.03 0.240.03 Unknown 0.58 0.04 0.58 0.09 0.58 0.07 0.58 0.12 0.57 0.04 0.58 0.070.58 0.06 Unknown 0.59 0.05 0.59 0.07 0.6 0.06 0.59 0.16 0.59 0.04 0.60.05 0.59 0.05 Unknown 0.62 0.04 0.62 0.07 0.62 0.06 0.62 0.22 0.62 0.040.62 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.03 0.81 0.02 0.81 0.07 — — — —— — Unknown 0.87 0.08 0.87 0.12 0.87 0.15 0.87 0.18 0.87 0.13 0.87 0.140.87 0.13 Unknown 0.89 0.03 0.89 0.12 0.89 0.04 0.89 0.52 0.89 0.04 0.890.02 0.89 0.02 Unknown 0.91 — — — — — 0.91 0.18 — — — — — — Unknown 0.95— — — — — 0.95 0.07 — — — — — — Unknown 1.03 0.19 1.02 0.07 1.03 0.031.03 0.05 1.02 0.08 1.02 0.04 1.03 0.09 Unknown 1.11 0.04 1.11 0.05 1.110.06 1.12 0.06 1.11 0.05 1.11 0.05 1.11 0.05 Unknown 1.27 0.28 1.27 0.411.27 0.32 1.27 0.19 1.27 0.38 1.27 0.27 1.27 0.61 Unknown 1.3 0.19 1.30.09 1.3 0.25 1.3 0.01 1.3 0.21 1.3 0.26 1.3 0.17 Unknown 1.39 0.08 1.390.05 1.39 0.08 1.39 0.03 1.39 0.7 1.39 0.06 1.39 0.08 Unknown 1.41 0.021.41 0.01 1.41 0.02 1.41 0 1.41 0.02 1.41 0.01 1.41 0.02 Unknown 1.460.14 1.46 0.06 1.46 0.08 1.46 0.06 1.46 0.09 1.46 0.08 1.46 0.12 Unknown1.72 — — — 1.72 0.07 1.73 0.07 — — 1.72 0.07 1.73 0.02 Unknown 2.31 0.042.31 0.04 2.3 0.04 2.31 0.03 2.32 0.04 2.3 0.04 2.31 0.08 Unknown 2.6 —2.6 0.03 2.59 0.04 2.6 0.02 2.6 0.05 2.59 0.03 2.6 0.06 Unknown 2.8 —2.8 0.03 2.79 0.03 2.82 0.03 2.81 0.03 2.79 0.03 2.8 0.04

TABLE 1.16 3,4,3-L1(1,2-HOPO)-CROSCARMELLOSE SODIUM COMPATIBILITY 40°C./75% RH 25° C./60% RH Test Initial (T = O) 2 Weeks 4 weeks 8 Weeks 2Weeks 4 weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellowPale Pale Pale Observation yellow pasty pasty pasty yellow yellow yellowpowder material material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3LIO) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3LIO Purity 98.75 98.77 98.69 98.43 98.77 98.74 98.58Total Impurities 1.25 1.23 1.31 1.57 1.23 1.26 1.42 RRT % w/w RRT % w/wRRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.06 0.24 0.04 0.24 0.05 0.24 0.03 0.24 0.04 0.240.04 Unknown 0.59 0.02 0.58 0.07 0.58 0.08 0.58 0.08 0.58 0.05 0.58 0.070.58 0.06 Unknown 0.6 0.02 0.59 0.05 0.6 0.05 0.59 0.06 0.59 0.04 0.60.05 0.59 0.05 Unknown 0.63 0.02 0.62 0.06 0.62 0.05 0.62 0.06 0.62 0.040.62 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.02 0.81 0.02 0.81 0.02 — — — —— — Unknown 0.87 0.07 0.87 0.13 0.87 0.15 0.87 0.16 0.87 0.13 0.87 0.150.87 0.15 Unknown 0.89 0.03 0.89 0.04 0.89 0.02 0.89 0.02 0.89 0.03 0.890.02 0.89 0.02 Unknown 1.03 0.21 1.03 0.08 1.02 0.04 1.03 0.11 1.03 0.081.03 0.04 1.03 0.09 Unknown 1.11 0.05 1.11 0.05 1.11 0.06 1.11 0.07 1.110.05 1.11 0.06 1.11 0.06 Unknown 1.26 0.31 1.27 0.31 1.27 0.23 1.27 0.351.27 0.29 1.27 0.2 1.27 0.35 Unknown 1.29 0.2 1.3 0.11 1.3 0.26 1.3 0.231.3 0.26 1.3 0.29 1.3 0.22 Unknown 1.38 0.08 1.39 0.03 1.39 0.04 1.390.04 1.39 0.03 1.39 0.03 1.39 0.03 Unknown 1.4 0.04 1.41 0.01 1.41 0.011.41 0.01 1.41 0.02 1.41 0.01 1.41 0.01 Unknown 1.45 0.14 1.46 0.09 1.460.08 1.46 0.08 1.47 0.05 1.46 0.06 1.46 0.07 Unknown 1.72 — — — 1.720.06 1.73 0.08 — — 1.72 0.09 1.73 0.06 Unknown 2.29 0.05 2.31 0.05 2.30.04 2.31 0.07 2.32 0.05 2.3 0.05 2.31 0.08 Unknown 2.6 — 2.6 0.03 2.590.03 2.6 0.04 2.61 0.04 2.59 0.03 2.6 0.04 Unknown 2.8 — 2.8 0.03 2.790.03 2.8 0.03 2.81 0.03 2.79 0.02 2.8 0.03

TABLE 1.17 3,4,3-LI(1,2-HOPO)-SODIUM STARCH GLYCOLATE COMPATIBILITY 40°C./75% RH 25°C./60% RH Test Initial (T = O) 2 Weeks 4 weeks 8 Weeks 2Weeks 4 weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellowPale Pale Pale Observation yellow pasty pasty pasty yellow yellow yellowpowder material material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3LIO) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3LIO Purity 98.77 98.79 98.69 98.53 98.81 98.77 98.55Total Impurities 1.23 1.21 1.31 1.47 1.19 1.23 1.45 RRT % w/w RRT % w/wRRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.07 0.24 0.1 0.24 0.06 0.24 0.03 0.24 0.04 0.240.04 Unknown 0.58 0.05 0.58 0.07 0.58 0.09 0.58 0.07 0.57 0.05 0.58 0.060.58 0.06 Unknown 0.59 0.04 0.59 0.06 0.6 0.06 0.59 0.05 0.59 0.04 0.60.04 0.59 0.04 Unknown 0.62 0.03 0.62 0.06 0.62 0.06 0.62 0.05 0.62 0.040.62 0.04 0.62 0.05 Unknown 0.81 — 0.81 0.02 0.81 0.03 0.81 0.02 — — — —— — Unknown 0.87 0.12 0.87 0.14 0.87 0.15 0.87 0.16 0.87 0.13 0.87 0.150.87 0.15 Unknown 0.89 0.03 0.89 0.07 0.89 0.12 0.89 0.02 0.89 0.03 0.890.02 0.89 0.02 Unknown 1.03 0.21 1.03 0.08 1.02 0.05 1.03 0.08 1.03 0.081.02 0.04 1.03 0.09 Unknown 1.11 0.04 1.11 0.05 1.11 0.06 1.11 0.07 1.110.05 1.11 0.06 1.11 0.06 Unknown 1.27 0.25 1.27 0.3 1.27 0.24 1.27 0.371.27 0.32 1.27 0.23 1.27 0.39 Unknown 1.3 0.25 1.3 0.11 1.3 0.1 1.3 0.21.3 0.25 1.3 0.27 1.3 0.23 Unknown 1.39 0.05 1.39 0.03 1.39 0.03 1.390.04 1.39 0.03 1.39 0.03 1.39 0.04 Unknown 1.41 0.01 1.41 0.01 1.41 01.41 0.01 1.41 0.02 1.41 0.01 1.41 0.01 Unknown 1.46 0.11 1.46 0.07 1.460.06 1.46 0.07 1.47 0.04 1.46 0.06 1.46 0.07 Unknown 1.72 — — — 1.720.08 1.72 0.09 — — 1.72 0.07 1.73 0.06 Unknown 2.31 0.04 2.31 0.04 2.30.03 2.31 0.06 2.32 0.04 2.3 0.04 2.31 0.07 Unknown 2.6 — 2.6 0.03 2.590.02 2.6 0.03 2.61 0.03 2.59 0.03 2.6 0.04 Unknown 2.8 — 2.8 0.03 2.790.02 2.8 0.03 2.81 0.02 2.79 0.03 2.8 0.03

TABLE 1.18 3,4,3-LI( 1,2-HOPO)-CROSPOVIDONE COMPATIBILITY 40° C./75% RH25° C./60% RH Test Initial (T = O) 2 Weeks 4 weeks 8 Weeks 2 Weeks 4weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellow Pale PalePale Observation yellow pasty pasty pasty yellow yellow yellow powdermaterial material material powder powder powder Identification RTmatches RT matches RTmatches RT matches RT matches RT matches RT matchesby HPLC with with with with with with with (3LIO) standard standardstandard standard standard standard standard Related Substances (% Area,n = 1) 3LIO Purity 98.74 98.49 98.57 98.47 98.74 98.74 98.52 TotalImpurities 1.26 1.51 1.43 1.53 1.26 1.26 1.48 RRT % w/w RRT % w/w RRT %w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown Impurities Unknown0.24 — 0.24 0.14 0.24 0.04 0.24 0.05 0.24 0.03 0.24 0.04 0.24 0.04Unknown 0.58 0.05 0.58 0.1 0.58 0.08 0.58 0.09 0.58 0.05 0.58 0.07 0.580.07 Unknown 0.59 0.04 0.59 0.08 0.6 0.06 0.59 0.07 0.59 0.05 0.6 0.050.59 0.05 Unknown 0.62 0.04 0.62 0.08 0.62 0.05 0.62 0.07 0.62 0.06 0.620.05 0.62 0.06 Unknown 0.81 — 0.81 0.06 0.81 0.02 0.81 0.02 — — — — — —Unknown 0.87 0.1 0.87 0.13 0.87 0.16 0.87 0.17 0.87 0.14 0.87 0.15 0.870.15 Unknown 0.89 0.03 0.89 0.24 0.89 0.03 0.89 0.03 0.89 0.04 0.89 0.030.89 0.02 Unknown 1.03 0.2 1.02 0.06 1.03 0.03 1.03 0.11 1.03 0.08 1.030.05 1.03 0.07 Unknown 1.11 0.04 1.13 0.1 1.11 0.06 1.11 0.05 1.11 0.051.11 0.06 1.11 0.06 Unknown 1.27 0.31 1.27 0.25 1.27 0.28 1.27 0.32 1.270.31 1.27 0.22 1.27 0.39 Unknown 1.3 0.19 1.3 0.11 1.3 0.25 1.3 0.24 1.30.23 1.3 0.26 1.3 0.24 Unknown 1.39 0.09 1.39 0.04 1.39 0.08 1.39 0.051.39 0.05 1.39 0.04 1.39 0.05 Unknown 1.41 0.02 1.41 0 1.41 0.01 1.41 01.41 0.01 1.41 0.01 1.41 0.01 Unknown 1.46 0.12 1.46 0.06 1.46 0.09 1.460.06 1.46 0.07 1.46 0.06 1.46 0.08 Unknown 1.72 — — — 1.72 0.09 1.720.11 — — 1.72 0.08 1.73 0.07 Unknown 2.31 0.04 2.31 0.03 2.3 0.04 2.310.06 2.32 0.04 2.3 0.03 2.31 0.07 Unknown 2.6 — 2.6 0.03 2.59 0.04 2.60.03 2.6 0.04 2.59 0.03 2.6 0.04 Unknown 2.8 — 2.8 0.03 2.79 0.03 2.80.02 2.81 0.03 2.79 0.03 2.8 0.03

TABLE 1.19 3,4,3-LI(1,2-HOPO)-COLLOIDAL SILICON DIOXIDE COMPATIBILITY40° C./75% RH 25° C./60% RH Test Initial (T = O) 2 Weeks 4 weeks 8 Weeks2 Weeks 4 weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellowPale Pale Pale Observation yellow pasty pasty pasty yellow yellow yellowpowder material material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3LIO) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3LIO Purity 98.77 98.57 98.52 98.39 98.70 98.65 98.29Total Impurities 1.23 1.43 1.48 1.61 1.30 1.35 1.71 RRT % w/w RRT % w/wRRT % w/w RRT %w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.14 0.24 0.04 0.24 0.06 0.24 0.03 0.24 0.03 0.240.04 Unknown 0.58 0.06 0.58 0.09 0.58 0.07 0.58 0.09 0.58 0.05 0.58 0.060.58 0.06 Unknown 0.59 0.04 0.59 0.08 0.6 0.06 0.59 0.06 0.59 0.04 0.60.05 0.59 0.05 Unknown 0.62 0.04 0.62 0.07 0.62 0.06 0.62 0.07 0.62 0.040.62 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.03 0.81 0.02 0.81 0.02 — — — —— — Unknown 0.87 0.11 0.87 0.12 0.87 0.15 0.87 0.16 0.87 0.12 0.87 0.130.87 0.13 Unknown 0.89 0.03 0.89 0.15 0.89 0.03 0.89 0.03 0.89 0.03 0.890.03 0.89 0.02 Unknown 1.03 0.2 1.03 0.06 1.02 0.03 1.03 0.11 1.02 0.071.02 0.04 1.02 0.07 Unknown 1.11 0.04 1.11 0.05 1.11 0.05 1.11 0.06 1.110.05 1.11 0.05 1.11 0.05 Unknown 1.27 0.25 1.27 0.39 1.27 0.34 1.27 0.411.27 0.4 1.27 0.34 1.27 0.66 Unknown 1.3 0.23 1.3 0.06 1.3 0.25 1.3 0.191.3 0.2 1.3 0.23 1.3 0.17 Unknown 1.39 0.07 1.39 0.03 1.39 0.09 1.390.07 1.39 0.05 1.39 0.07 1.39 0.08 Unknown 1.41 0.01 1.41 0.01 1.41 0.021.41 0.01 1.41 0.03 1.41 0.02 1.41 0.02 Unknown 1.46 0.12 1.46 0.06 1.460.1 1.46 0.08 1.46 0.07 1.46 0.1 1.46 0.12 Unknown 1.72 — — — 1.72 0.061.72 0.08 — — 1.72 0.04 1.73 0.02 Unknown 2.31 0.04 2.31 0.04 2.3 0.042.31 0.06 2.32 0.04 2.3 0.05 2.31 0.08 Unknown 2.6 — 2.6 0.03 2.59 0.042.6 0.04 2.6 0.05 2.59 0.05 2.6 0.07 Unknown 2.8 — 2.8 0.03 2.79 0.032.8 0.02 2.81 0.02 2.79 0.03 2.8 0.04

TABLE 1.20 3,4,3-LI(1,2-HOPO)-MAGNESIUM STEARATE COMPATIBILITY 40°C./75% RH 25° C./60% RH Test Initial (T = O) 2 Weeks 4 weeks 8 Weeks 2Weeks 4 weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellowPale Pale Pale Observation yellow pasty pasty pasty yellow yellow yellowpowder material material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3LIO) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3LIO Purity 98.75 98.67 98.41 98.47 98.68 98.70 98.37Total Impurities 1.25 1.33 1.59 1.53 1.32 1.30 1.63 RRT % w/w RRT % w/wRRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.04 0.24 0.15 0.24 0.06 0.24 0.03 0.24 0.03 0.240.04 Unknown 0.58 0.06 0.58 0.06 0.58 0.1 0.58 0.09 0.58 0.05 0.58 0.060.58 0.06 Unknown 0.59 0.05 0.59 0.05 0.6 0.08 0.59 0.06 0.59 0.04 0.60.05 0.59 0.05 Unknown 0.62 0.04 0.62 0.05 0.62 0.08 0.62 0.08 0.62 0.040.62 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.02 0.81 0.03 0.81 0.02 — — — —— — Unknown 0.87 0.11 0.87 0.14 0.87 0.13 0.87 0.16 0.87 0.13 0.87 0.130.87 0.14 Unknown 0.89 0.03 0.89 0.03 0.89 0.21 0.89 0.03 0.89 0.03 0.890.02 0.89 0.02 Unknown 1.03 0.21 1.03 0.07 1.02 0.03 1.03 0.1 1.02 0.081.02 0.03 1.03 0.08 Unknown 1.11 0.04 1.13 0.05 1.12 0.07 1.11 0.06 1.110.05 1.11 0.05 1.11 0.05 Unknown 1.27 0.27 1.27 0.32 1.27 0.35 1.27 0.341.27 0.34 1.27 0.32 1.27 0.52 Unknown 1.3 0.21 1.3 0.23 1.3 0.06 1.30.18 1.3 0.23 1.3 0.22 1.3 0.2 Unknown 1.39 0.07 1.39 0.06 1.39 0.061.39 0.06 1.39 0.07 1.39 0.07 1.39 0.09 Unknown 1.41 0.01 1.41 0.02 1.410.01 1.41 0.01 1.41 0.02 1.41 0.02 1.41 0.01 Unknown 1.46 0.13 1.46 0.061.46 0.08 1.46 0.06 1.46 0.07 1.46 0.09 1.46 0.11 Unknown 1.72 — — —1.72 0.06 1.72 0.1 — — 1.72 0.03 1.73 0.04 Unknown 2.31 0.04 2.31 0.062.3 0.03 2.31 0.06 2.32 0.05 2.3 0.04 2.31 0.07 Unknown 2.6 — 2.6 0.052.59 0.03 2.6 0.03 2.6 0.05 2.59 0.06 2.6 0.05 Unknown 2.8 — 2.8 0.042.79 0.02 2.8 0.02 2.81 0.03 2.79 0.03 2.8 0.03

TABLE 1.21 3,4,3-LI(1,2-HOPO)-HYDROGENATED VEGETABLE OIL COMPATIBILITY40° C./75% RH 25° C./60% RH Test Initial (T = 0) 2 Weeks 4 weeks 8 Weeks2 Weeks 4 weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellowPale Pale Pale Observation yellow pasty pasty pasty yellow yellow yellowpowder material material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3L1O) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3L1O 98.73 98.58 98.37 96.98 98.71 98.65 98.39 PurityTotal 1.27 1.42 1.63 3.02 1.29 1.35 1.61 Impurities RRT % w/w RRT % w/wRRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.14 0.24 0.24 0.24 0.48 0.24 0.03 0.24 0.04 0.240.04 Unknown 0.58 0.05 0.58 0.1 0.58 0.14 0.58 0.24 0.58 0.06 0.58 0.070.58 0.07 Unknown 0.59 0.04 0.59 0.08 0.6 0.1 0.59 0.17 0.59 0.05 0.60.05 0.59 0.05 Unknown 0.62 0.04 0.62 0.08 0.62 0.12 0.62 0.2 0.62 0.050.62 0.05 0.62 0.05 Unknown 0.81 — 0.81 0.03 0.81 0.05 0.81 0.12 — — — —— — Unknown 0.87 0.1 0.87 0.12 0.87 0.12 0.87 0.2 0.87 0.14 0.87 0.140.87 0.14 Unknown 0.89 0.03 0.89 0.14 0.89 0.28 0.89 0.71 0.89 0.03 0.890.02 0.89 0.02 Unknown 0.91 — — — — — 0.91 0.24 — — — — — — Unknown 0.95— — — — — 0.95 0.17 — — — — — — Unknown 1.03 0.2 1.02 0.05 1.02 0.021.03 0.04 1.03 0.07 1.02 0.03 1.03 0.08 Unknown 1.11 0.03 1.11 0.06 1.120.09 1.12 0.14 1.11 0.05 1.11 0.05 1.11 0.06 Unknown 1.27 0.31 1.27 0.351.27 0.17 1.27 0.06 1.27 0.29 1.27 0.29 1.27 0.5 Unknown 1.3 0.2 1.30.07 1.3 0.04 1.3 0.01 1.3 0.24 1.3 0.24 1.3 0.2 Unknown 1.39 0.09 1.390.03 1.39 0.04 1.39 0.05 1.39 0.08 1.39 0.1 1.39 0.08 Unknown 1.41 0.021.41 0.01 1.41 0 1.41 0 1.41 0.02 1.41 0.2 1.41 0.01 Unknown 1.46 0.111.46 0.06 1.46 0.08 1.46 0.07 1.46 0.07 1.46 0.09 1.46 0.11 Unknown 1.72— — — 1.72 0.07 1.72 0.07 — — 1.72 0.05 1.73 0.04 Unknown 2.31 0.04 2.310.04 2.3 0.03 2.31 0.02 2.31 0.04 2.3 0.05 2.31 0.08 Unknown 2.6 — 2.60.03 2.59 0.03 2.59 0.02 2.6 0.03 2.59 0.05 2.6 0.06 Unknown 2.8 — 2.80.03 2.79 0.02 2.8 0.02 2.81 0.03 2.79 0.03 2.8 0.04

TABLE 1.22 3,4,3-LI(1,2-HOPO)-POLYSORBATE 80 COMPATIBILITY 40° C./75% RH25° C./60% RH Test Initial (T = 0) 2 Weeks 4 weeks 8 Weeks 2 Weeks 4weeks 8 Weeks Visual Pale Pale yellow Pale yellow Pale yellow Pale PalePale Observation yellow pasty pasty pasty yellow yellow yellow powdermaterial material material powder powder powder Identification RTmatches RT matches RT matches RT matches RT matches RT matches RTmatches by HPLC with with with with with with with (3L1O) standardstandard standard standard standard standard standard Related Substances(% Area, n = 1) 3L1O 98.76 98.63 98.43 98.14 98.57 98.54 98.46 PurityTotal 1.24 1.37 1.57 1.86 1.43 1.46 1.54 Impurities RRT % w/w RRT % w/wRRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT % w/w Unknown ImpuritiesUnknown 0.24 — 0.24 0.05 0.24 0.19 0.24 0.14 0.24 0.03 0.24 0.03 0.240.05 Unknown 0.58 0.05 0.58 0.07 0.58 0.16 0.58 0.13 0.58 0.05 0.58 0.070.58 0.07 Unknown 0.59 0.04 0.59 0.05 0.6 0.14 0.59 0.1 0.59 0.05 0.60.05 0.59 0.06 Unknown 0.62 0.04 0.62 0.06 0.62 0.12 0.62 0.11 0.62 0.040.62 0.05 0.62 0.06 Unknown 0.81 — 0.81 0.02 0.81 0.07 0.81 0.06 — — — —— — Unknown 0.87 0.12 0.87 0.13 0.87 0.14 0.87 0.19 0.87 0.11 0.87 0.150.87 0.14 Unknown 0.89 0.03 0.89 0.04 0.89 0.19 0.89 0.22 0.89 0.03 0.890.06 0.89 0.04 Unknown 0.91 — — — — — 0.91 0.07 — — — — — — Unknown 1.030.19 1.02 0.07 1.03 0.04 1.02 0.09 1.02 0.08 1.02 0.03 1.03 0.08 Unknown1.11 0.04 1.11 0.06 1.12 0.12 1.12 0.08 1.11 0.05 1.11 0.05 1.11 0.06Unknown 1.27 0.24 1.27 0.35 1.27 0.1 1.27 0.38 1.27 0.45 1.27 0.32 1.270.45 Unknown 1.3 0.24 1.3 0.19 1.3 0.04 1.3 0.02 1.3 0.19 1.3 0.27 1.30.18 Unknown 1.39 0.08 1.39 0.07 1.39 0.04 1.39 0.04 1.39 0.08 1.39 0.081.39 0.06 Unknown 1.41 0.01 1.41 0.02 1.41 0 1.41 0 1.41 0.04 1.41 0.021.41 0.01 Unknown 1.46 0.11 1.46 0.08 1.46 0.05 1.46 0.06 1.46 0.11 1.460.1 1.46 0.08 Unknown 1.72 — — — 1.72 0.14 1.73 0.15 — — 1.72 0.06 1.730.07 Unknown 2.31 0.04 2.31 0.05 2.3 0.02 2.31 0.03 2.31 0.05 2.3 0.042.31 0.06 Unknown 2.6 — 2.6 0.04 2.59 0.01 2.6 0.01 2.6 0.05 2.59 0.042.6 0.04 Unknown 2.8 — 2.8 0.03 2.79 0.01 2.8 0.01 2.8 0.03 2.79 0.032.8 0.03

TABLE 1.23 3,4,3-LI(1,2-HOPO) CONTROL 40° C./75% RH 25° C./60% RH TestInitial (T = 0) 2 Weeks 4 weeks 8 Weeks 2 Weeks 4 weeks 8 Weeks VisualPale Pale yellow Pale yellow Pale yellow Pale Pale Pale Observationyellow pasty pasty pasty yellow yellow yellow powder material materialmaterial powder powder powder Identification RT matches RT matches RTmatches RT matches RT matches RT matches RT matches by HPLC with withwith with with with with (3L1O) standard standard standard standardstandard standard standard Related Substances (% Area, n = 1) 3L1O 98.7398.62 98.63 97.49 98.67 98.66 98.47 Purity Total 1.27 1.38 1.37 2.511.33 1.34 1.53 Impurities RRT % w/w RRT % w/w RRT % w/w RRT % w/w RRT %w/w RRT % w/w RRT % w/w Unknown Impurities Unknown 0.24 — 0.24 0.06 0.240.05 0.24 0.34 0.24 0.03 0.24 0.03 0.24 0.04 Unknown 0.58 0.05 0.58 0.070.58 0.09 0.58 0.2 0.58 0.05 0.58 0.07 0.58 0.07 Unknown 0.59 0.04 0.590.06 0.6 0.06 0.59 0.16 0.59 0.05 0.6 0.05 0.59 0.05 Unknown 0.62 0.040.62 0.06 0.62 0.07 0.62 0.18 0.62 0.05 0.62 0.05 0.62 0.06 Unknown 0.81— 0.81 0.02 0.81 0.02 0.81 0.05 — — — — — — Unknown 0.87 0.1 0.87 0.130.87 0.16 0.87 0.18 0.87 0.12 0.87 0.14 0.87 0.15 Unknown 0.89 0.02 0.890.07 0.89 0.03 0.89 0.32 0.89 0.03 0.89 0.02 0.89 0.02 Unknown 0.91 — —— — — 0.91 0.11 — — — — — — Unknown 0.95 — — — — — 0.95 0.04 — — — — — —Unknown 1.03 0.21 1.02 0.07 1.02 0.03 1.02 0.09 1.02 0.08 1.03 0.04 1.030.08 Unknown 1.11 0.04 1.11 0.06 1.11 0.06 1.12 0.05 1.11 0.05 1.11 0.051.11 0.05 Unknown 1.27 0.29 1.27 0.36 1.27 0.26 1.27 0.4 1.27 0.36 1.270.28 1.27 0.41 Unknown 1.3 0.21 1.3 0.16 1.3 0.2 1.31 0.01 1.3 0.22 1.30.26 1.3 0.23 Unknown 1.39 0.09 1.39 0.05 1.39 0.74 1.39 0.08 1.39 0.071.39 0.07 1.39 0.07 Unknown 1.41 0.01 1.41 0.01 1.41 0.01 1.41 0 1.410.03 1.41 0.01 1.41 0.01 Unknown 1.46 0.11 1.46 0.07 1.46 0.08 1.46 0.121.46 0.08 1.46 0.09 1.46 0.09 Unknown 1.72 — — — 1.72 0.09 1.72 0.1 — —1.72 0.05 1.73 0.06 Unknown 2.31 0.05 2.31 0.05 2.3 0.04 2.31 0.03 2.310.05 2.3 0.05 2.31 0.07 Unknown 2.59 — 2.59 0.04 2.59 0.03 2.6 0.02 2.60.04 2.59 0.04 2.6 0.05 Unknown 2.8 — 2.8 0.03 2.79 0.02 2.8 0.02 2.80.03 2.79 0.03 2.8 0.036. Conclusion

A series of commonly used pharmaceutical excipients were tested forinteractions and compatibility with 3,4,3-L1(1,2-HOPO). Among those 14compounds tested, 4 excipients (pregelatinized starch, compressiblesugar, providone, and hydrogenated vegetable oil) resulted in a decreasein 3,4,3-L1(1,2-HOPO) purity or in an increase of specific impuritycontent. Those 4 excipients should be avoided in future formulations of3,4,3-L1(1,2-HOPO).

Example 2—Feasibility of Developing Oral Formulations for3,4,3-L1(1,2-HOPO) Summary

The feasibility of developing oral formulations for 3,4,3-L1(1,2-HOPO)was evaluated. Four oral dosage forms were investigated: (i) powder inbottle, (ii) dispersible/dissolvable granules, (iii) chewable tablets,and (iv) conventional immediate release tablets. Based on the studiesperformed, nine formulation prototypes that showed immediate drugrelease behavior and required physical properties were identified andselected for API verification, gastric fluid dissolution, and relatedsubstance testing following defined liquid chromatography methods. Amongthese selected compositions, two are powder in bottle formulations, twoare granule formulations, three are chewable tablet formulations, andtwo are conventional tablet formulations. The respective compositions ofthese prototype formulations are summarized and tabulated in TABLE 2.1.A11 assays confirmed that these prototypes are suitable for furtherdevelopment. The stability of these formulations will be evaluated priorto a first-in-human trial for 3,4,3-L1(1,2-HOPO). These stabilitystudies will also include capsules containing the powder in bottlecomposition A2, which may be the optimal dosage form for adjusting doeslevels in clinical settings.

TABLE 2.1 COMPOSITIONS OF PROTOTYPE FORMULATIONS Conventional DosagePowder in Granules Chewable Tables Tables Ingredients Form → A2 A11 G11G12 C11 C13 C21 T50 T51 Intra-Granular Materials (for granules andconventional tablets) 3,4,3-L1(1,2-HOPO) 1.000 1.000 1.000 1.000 0.5000.500 0.500 0.500 0.500 Sodium Oleate 0.092 0.092 0.092 0.092 0.0460.046 0.046 0.046 0.046 Microcrystalliine — 1.000 — — — — — — Celluloseand Carboxymethyl Cellulose NF (Avivel RC-591) Croscarmellose — — 0.0750.075 0.075 — 0.075 0.084 0.092 Sodium, NF (Ac-Di-Sol) Microcrystalliine— — 1.833 — 1.854 — 0.927 — — Cellulose and guar gum, NF (Avivel Lactose— 1.533 — — — — — — Monohydrate, NF (Pharmatose 300 M) Lactose — — — — —1.929 — — — Monohydrate, Povidone and Crospovidone, NF (Ludipress)Mannitol, USP — — — — — — 0.9227 — — (Mannogem) Magensium sterate, — — —— 0.025 0.025 0.025 — — NF (HyQual) Microcrystalliine — — — — — — —0.410 0.501 Cellulose and guar gum, NF (Avivel Colloidal silicone — — —— — — — 0.005 0.006 dioxide, NF (Cab-O-Sil M5P) Purified water USP — —Q.S. Q.S. — — — — — Magensium sterate, — — — — — — — NF (HyQual) ExtraGranular Materials (for granules and conventional tablets)Hvnromellose.50 cps — — — 0.300 — — — — Magnesium sterate, — — — — — — —0.005 0.006 NF (HyQual) Unit weight (g) 1.092 2.0962 3.000 3.000 2.5002.500 2.500 1.050 1.1511. Purpose of Study

The purpose of this study was to provide data that can be used tosupport research efforts. It was not conducted in accordance with U.S.Food and Drug Administration (FDA) “Good Laboratory Practice forNonclinical Laboratory Studies” (GLP) regulations, as described in 21CFR Part 58. However, the study was planned, performed, recorded, andreported in accordance with standard practices to ensure data qualityand integrity.

2. Objective of Study

The objective of this study was to develop prototype oral formulationsof the active pharmaceutical ingredient 3,4,3-L1(1,2-HOPO). The clinicaldose of 3,4,3-L1(1,2-HOPO) is expected to be in the range of 1-2 gramsper unit. In order to retain the flexibility of dosing lower and higherdose strengths in clinical evaluation, several oral formulations wereincluded in the development work, including:

-   -   Powder in bottle (PIB)    -   Orally dispersible/dissolvable granules    -   Chewable tablets    -   Conventional oral tablets        3. Experimental Design

Suitable excipients were selected based on the results of drug-excipientcompatibility studies (3,4,3-L1(1,2-HOPO)—Excipient Compatibility Study;EXAMPLE 1), and evaluated for feasibility of developing the selectedformulations. A11 test formulations contained sodium oleate as apermeation enhancer, based on pharmacokinetic results established inparallel. In addition to 3,4,3-L1(1,2-HOPO), diluents, and thepermeation enhancer, other formulation components were also probed foreach prototype formulation. A typical formulation matrix is shown inTABLE 2.2.

TABLE 2.2 FORMULATION MATRIX Formulation Typical Composition Component 12 3 4 API 3,4,3-LI(1,2-HOPO) (20-80%) Permeation Sodium Oleate enhancer(1- Note: Oleic acid is being used in oral drug products (max potency:598 mg). The sodium 10%) salt of oleic acid has not been reported inapproved oral drug products, however, literature reports support its useas a permeation enhancer for oral applications. References:http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm; Pharm. Res.25, 8, (2008). Diluent Coprocessed Coprocessed Lactose MicrocrystallineLactose (10-70%) Microcrystalline Monohydrate and Cellulose, NFMonohydrate, NF Cellulose and guar Povidone (Example: (Example: Avicel(Pharmatose 350 Gum (Example: Ludipress from BASF) PH 101/Avicel PHM/SuperTab 11SD Avicel CE-15 from 102 from FMC from FMC Biopolymer)Biopolymer) DMV - Fonterra) Binders PVP/HPMC PVP/HPMC PVP/HPMC PVP/HPMC(1-6%) Disintegrants Croscarmellose Sodium, Crospovidone, Sodium StarchGlycolate (2-8%) Lubricants & Colloidal Silicon Dioxide, Magnesiumstearate glidants (0.2-20%)

Suitable formulation methodologies such as direct compression, drycompaction, and/or wet granulation processes were evaluated. Selectedprototype compositions were tested for various physicochemicalproperties as shown in TABLE 2.3.

TABLE 2.3 PHYSICOCHEMICAL PROPERTIES TESTED Evaluation Sample No. DosageForm Granular Properties Tables Properties 1 Powder in bottle 1.Appearance Not Applicable 2. Fines to Course ration 2 Orallydispersible/ 3. Loss on drying dissolvable 4. Flow nature granules 5.Bulk and tapped densities 6. Dispersibility in water 7. Dissolutionassay 8. Interference assay 9. Filter adsorption assay 3 Chewabletables 1. Appearance 1. Appearance. 2. Flow nature 2. Weight 4Conventional oral 3. Compressibility index 3. Thickness. tables 4.Hardness 5. Friability 6. Disintegration Time 7. Dissolution assay 8.Interference assay 9. Filter adsorption assay4. Materials And Methods

a. Test and Control Articles

Test Article: 3,4,3-L1(1,2-HOPO)

Manufacturer: Ash Stevens, Inc. (Detroit, Mich.)

Lot Number: ML-11-276

Physical Description: Pale yellow solid

Storage Conditions: Refrigerated 2-8° C. protected from light.

Analytical Materials:

Purified Water, USP HPLC Grade - Supplier: Ricca Chemical Inc.Trifluoroacetic acid ACS Grade - Supplier: Sigma Aldrich Formic AcidHPLC Grade - Supplier: EMD Chemicals Acetonitrile HPLC Grade - Supplier:Fischer Scientific HPLC Column Agilent, Eclipse XDB-C18, 4.6 × 150 mm, 5μmFormulation Components:

-   -   Croscarmellose Sodium, NF, Ph. Eur., JP (Ac-Di-Sol)    -   FMC Biopolymer, Lot #TN13825327    -   Crospovidone, NF, Ph. Eur., JPE (Kollidone-CLM)    -   BASF, Lot #10204988Q0    -   Sodium Starch Glycolate, NF    -   Spectrum Chemicals, Lot #1BC0437    -   Lactose Monohydrate, USP/NF, Ph. Eur., JP (SuperTab 11SD)    -   DFE Pharma, Lot #10697993/5731011    -   Lactose Monohydrate, USP/NF, Ph. Eur., JP (Pharmatose 300M)    -   DFE Pharma, Lot #10601833/9445861    -   Co-Processed Lactose monohydrate, povidone and crospovidone, NF        (Ludipress)    -   BASF, Lot #05266375L0    -   Microcrystalline cellulose, NF (Avicel PH102)    -   FMC Biopolymer, Lot #P212824001    -   Co-Processed microcrystalline cellulose and guar gum, GRAS        (Avicel CE-15)    -   FMC Biopolymer, Lot #RH10821854    -   Co-Processed microcrystalline cellulose and Carboxymethyl        Cellulose, NF (Avicel RC-591)    -   FMC Biopolymer, Lot #DN008820108    -   Povidone, USP (Plasdone K-29/32)    -   ISP Technologies, Lot #052304677    -   Mannitol, USP (Mannogem)    -   SPI Pharma, Lot #12000076G    -   Maltodextrin, NF (Glucidex IT 19)    -   Grain Processing Corporation, Lot #3084    -   Colloidal Silicon Dioxide (Cab-O-Sil MSP)    -   Cabot, Lot #3367714    -   Hypromellose, USP, 50 mPa·S    -   Spectrum Chemicals, Lot #1BJ2114    -   Sodium Oleate    -   Tokyo Chemical Industries Co. Ltd., Lot #3CSSIBI    -   Magnesium stearate, NF (HyQual)    -   Mallinckrodt, Lot #0912000002        A Prototype Preparation and Physico-Chemical Characterization

TABLE 2.4 PROTOTYPE PREPARATION AND PHYSICO- CHEMICAL CHARACTERIZATIONPowder in The API and other excipients, except sodium oleate, wereBottle, passed through sieve #30 and mixed with sodium oleate 1000 mg:(screened through mesh #40) following a geometrical addition approach.The blends were packed in glass vials (protected from light), labeled,and evaluated for the following properties: appearance, flow nature,bulk density, fines to coarse ratio, and dispersibility in water.Selected blends were evaluated for dissolution in simulated gastricfluid without enzymes, API content verification, and related substances.Granules, Granules were prepared by wet granulation process, 1000 mg:involving the following steps: (1) Sieving of the API and excipientsthrough sieve #30. (2) Preparation of granulation fluid solution intrials where binder such as povidone and hypromellose otherwise water isused as granulating fluid. (3) Mix API and excipients geometricallyusing plastic spatula. (4) Granulate the blend in stainless steel vesselusing plastic spatula. (5) Pass the wet granules through sieve #14 andallow it to dry in hot air oven at 60° C. until the LOD reaches below2%. (6) Pass the dried granules though sieve #14. (7) Sieve theextragranular materials through screen #20, and blend geometricallyusing plastic spatula. Lubricate the granules if necessary usingmagnesium stearate. The granules were packed in aluminum pouches(protected from light), labeled, and evaluated for the followingproperties: appearance, flow nature, fines to coarse ratio,dispersibility in water, and texture. Selected formulations wereevaluated for dissolution in simulated gastric fluid without enzymes,API content verification, and related substances. Chewable The chewabletablets were prepared by direct Tablets, compression process, involvingthe following steps: (1) 500 mg: Sieving of the API and excipients(except sodium oleate and magnesium stearate) through sieve #30. (2)Mixing of excipients and API manually. (3) Sieving of sodium oleate, andmagnesium stearate through mesh #40. (4) Mixing of sieved sodium oleatewith the API-excipient(s) blend. (5) Lubrication of the powder blendwith sieved magnesium stearate. (6) Evaluation of the physical blendsfor appearance, flow nature, and compressibility. (7) Compression of thepowder blends into tablets using suitable tooling. The tablets werepackaged in aluminum pouches (protected from light), labeled, andevaluated for the following properties: appearance, size, hardness,friability, thickness, disintegration time. Selected tablets wereevaluated for dissolution in simulated gastric fluid without enzymes,API content verification, and related substances. ConventionalFormulation of tablets by direct compression (DC) Tablets, involved thefollowing steps: (1) Sieving of the API and 500 mg: intragranularexcipients (except magnesium stearate) through sieve #30. (2) Mixing ofexcipients and API manually. (3) Sieving of magnesium stearate throughmesh #40. (4) Lubrication of the powder blend with sieved magnesiumstearate. (5) Evaluation of the physical blend for appearance, flownature, and compressibility. (6) Compression of the powder blend intotablets using suitable tooling. Formulation of tablets by wetgranulation (WG) involved the following steps: (1) Sieving of the APIand intra-granular excipients through sieve #30. (2) Mixing ofintragranular excipients and API manually. (3) Wet granulation of thepowder blend using purified water. (4) Drying of the wet granules at 60°C. until the loss on drying reaches <2.0%. (5) Screening of the driedgranules through mesh #14. (6) Sieving of magnesium stearate throughmesh #40. (7) Lubrication of the granules with sieved magnesiumstearate. (8) Evaluation of the physical blend for appearance, flownature, and compressibility. (9) Compression of the powder blend intotablets using suitable tooling. The tablets were packaged in glass vials(protected from light), labeled, and evaluated for the followingproperties: appearance, size, hardness, friability, thickness,disintegration time Selected tablets were evaluated for dissolution insimulated gastric fluid without enzymes, API content verification, andrelated substances. c. Sample Preparation for Solution Assays APIContent For powder in bottle, granules, and chewable tabletsVerification: crushed with a pestle and mortar, an amount of blendequivalent to 50 mg of 3,4,3-LI(1,2-HOPO) was weighed and transferredaccurately into a clean dry 100 mL volumetric flask. About 50 mL ofdiluent was added, mixed well and made up to the 100 mL mark. The flaskwas sonicated for about 30 minutes in ice water. For immediate releasetablets, 5 tablets were added to a clean dry 1000 mL volumetric flask.About 500 mL of diluent was added, mixed well and sonicated for 90minutes in ice water with intermittent shaking. The volume was made upto 1000 mL mark and further sonicated under ice for about 30 minutes. Inall cases, the sample solution was filtered through a 0.45μ Nylonsyringe filter and the filtrate was used for assay. The3,4,3-LI(1,2-HOPO) concentration in the sample solution was about 0.5mg/mL. Note: Throughout the sample preparation and storage, the flaskswere covered with aluminum foil. The samples were placed at 5° C. in theHPLC auto-sampler just after preparation to avoid any degradation, andthe run time was increased to 12 minutes to allow for columnequilibration. Dissolution The dissolution testing was performed in 900mL of Testing: simulated gastric fluid without enzymes maintained at 37± 0.5° C. using USP Apparatus II (Paddle) at 50 rpm. For each prototypeformulation, a unit dose was added to each of 6 dissolution vessels.Aliquots of 5 mL of sample were manually withdrawn and filtered througha 0.45μ Nylon syringe filter at required time intervals. Aliquots of 5mL of dissolution medium were replaced after sampling at each respectivetime point. The 3,4,3-LI(1,2- HOPO) concentration in the sample solutionwas about 1.1 mg/mL. Note: Throughout the sample preparation, thedissolution vessels were covered with aluminum foil. The samples wereplaced at 5° C. in the HPLC autosampler just after preparation to avoidany degradation, and the run time was increased to 12 minutes to allowfor column equilibration. Related For powder in bottle and granuleprototype formulations, Substances: one unit dose was transferred into aclean dry 250 mL volumetric flask. For tablet formulations, two tabletswere crushed and transferred into a clean dry 250 mL volumetric flask.In all cases, about 200 mL of diluent was added to the flask, which wasshaken in a wrist action shaker till a complete uniform dispersion wasobtained (about 40 minutes). The volume was made up to the mark withdiluent and mixed well. The sample solution was filtered using a 0.45μNylon syringe filter. Aliquots of 1 mL of the filtrate were diluted to 4mL in a scintillation vial and chromatographed. The 3,4,3-LI(1,2- HOPO)concentration in the sample solution was about 1.0 mg/mL. Note:Throughout the sample preparation and storage, the flasks were coveredwith aluminum foil. a. Chromatographic Assay and Purity AssessmentStandard Stock For each standard stock solutions, the test article wasSolutions: weighed (200 mg) and dissolved by sonication into 30 mL ofdiluent (water:acetonitrile = 90%:10%). After equilibration at roomtemperature, the volume of the standard solution was adjusted to 50 mL.Standard stock solutions were prepared in duplicates, working standardsolutions were prepared by dilution of each stock with the diluent tothe desired concentrations. Calibration With each experiment, 5calibration standard solutions at Standards: different concentrationswere prepared from stock solutions using the diluent. Concentrations ofthe calibration standards were between 0.2 and 2.0 mg/mL. Thecalibration standard solutions were chromatographed to demonstrate thelinearity of the calibration curve over the concentration range.Analytical Method I for Related Substance Assay (TABLE 2.5):

This method was previously established and validated (see3,4,3-L1(1,2-HOPO)—Excipient Compatibility Study; EXAMPLE 1).Suitability was therefore not re-evaluated as part of this study.

Instrument: Waters Alliance 2695 liquid chromatography system

Detector: 2487 Waters Dual Wavelength Detector

Column: Agilent, Eclipse XDB-C18, 4.6×150 mm, 5

Mobile Phase A: 0.05% formic acid in 95% H2O: 5% ACN

Mobile Phase B: 0.05% formic acid in acetonitrile (ACN)

Column Temperature: 25° C.

Flow Rate: 1.0 mL/min.

Injection Volume: 20 μL

Detection: 250 nm

Run Time: 50 min

Diluent: 9:1 H₂O:ACN

TABLE 2.5 GRADIENT CONDITIONS Time (min) A % B % 0.00 100 0 30.00 60 4040.00 0 100 41.00 100 0 50.10 100 0Analytical Method I for Related Substance Assay (TABLE 2.6):

This method was previously established and validated (see3,4,3-L1(1,2-HOPO)—Excipient Compatibility Study; EXAMPLE 1).Suitability was therefore not re-evaluated as part of this study.

Instrument: Waters Alliance 2695 liquid chromatography system

Detector: 2487 Waters Dual Wavelength Detector

Column: Agilent, Eclipse XDB-C18, 4.6×150 mm, 5 μm.

Mobile Phase A: 0.05% formic acid in 95% H2O: 5% ACN

Mobile Phase B: 0.05% formic acid in acetonitrile (ACN)

Column Temperature: 25° C.

Flow Rate: 1.0 mL/min.

Injection Volume: 20 μL

Detection: 250 nm

Run Time: 50 min

Diluent: 9:1 H₂O:ACN

TABLE 2.6 GRADIENT CONDITIONS Time (min) A % B % 0.00 100 0 30.00 60 4040.00 0 100 41.00 100 0 50.10 100 0Analytical Method II for API Content and Dissolution Assays (TABLE 2.7and TABLE 2.8):

TABLE 2.7 Instrument: Waters Alliance 2695 liquid chromatography systemDetector: 2487 Waters Dual Wavelength Detector Column: Waters, SymmetryC18, 2.1 × 150 mm, 5 μm. Mobile Phase A: 0.1% trifluoroacetic acid inH2O Mobile Phase B: 0.1% trifluoroacetic acid in ACN Column 30° C.Temperature: Flow Rate: 0.5 mL/min. Injection Volume: 10 μL Detection:250 nm Run Time: 10 min Diluent: 9:1 H₂O:ACN or simulated gastric fluidwithout enzymes, USP

TABLE 2.8 GRADIENT CONDITIONS Time (min) A % B % 0.00 90 10 3.5 58 424.5 58 42 5.0 2 98 6.5 2 98 7.0 90 10 10.0 90 10

-   Method II—Suitability Requirements: There should be no interference    from the diluent/blank at the retention times of 3,4,3-L1(1,2-HOPO)    peaks. The relative standard deviation (% RSD) for five replicate    system suitability injections should be below 2.0%. The response    factor of recovery check standards should be within 95-105%.-   Method II—Diluent Interference: There was no interference observed    from the API verification assay diluent (9:1 water:ACN) and    dissolution diluent (Simulated gastric fluid without enzymes, USP)    at the retention times of 3,4,3-L1(1,2-HOPO). The chromatograms of    API verification assay diluent and dissolution assay diluent are    shown in FIG. 3 and FIG. 4 , respectively. The chromatograms of    3,4,3-L1(1,2-HOPO) in API verification assay diluent and dissolution    assay diluent are shown in FIG. 5 and FIG. 6 , respectively.-   Method II—System Suitability: The RSDs of the five standard    injections were found to be less than 2.0% for both verification and    dissolution assays. The recoveries of the check standards were found    to be between 98.0% and 102% for both verification and dissolution    assays. The results of the system suitability evaluation are shown    in TABLE 2.9 and TABLE 2.10.

TABLE 2.9 3,4,3-LI(1,2-HOPO) VERIFICATION ASSAY - SYSTEM SUITABILITYEVALUATION Weight (mg)/Concentration 3,4,3-Li(1,2-HOPO) Details (mg/mL)Peak Area Standard inj#1 25.36/0.51 3915776 Standard inj#2 3983634Standard inj#3 3980352 Standard inj#4 3989295 Standard inj#5 4001175Average 3974046 SD 33524.63 % RSD 0.84 Limit % RSD NMT 2.0 Result PassCheck Standard inj#1 24.75/0.50 3826628 Check Standard inj#2 3910821Average 3868725 Check Standard Recovery % 100.3 Limit % Recovery98.0-102.0 Result Pass

TABLE 2.10 3,4,3-LI(1,2-HOPO) DISSOLUTION ASSAY - SYSTEM SUITABILITYEVALUATION Weight (mg)/Concentration 3,4,3-Li(1,2-HOPO) Details (mg/mL)Peak Area Standard inj#1 28.02/1.12 8366617 Standard inj#2 8587201Standard inj#3 8666099 Standard inj#4 8676613 Standard inj#5 8630739Average 8585454 SD 127236.98 % RSD 1.48 Limit % RSD NMT 2.0 Result PassCheck Standard inj#1 27.30/1.09 8264625 Check Standard inj#2 8401828Average 8333227 Check Standard Recovery % 100.4 Limit % Recovery98.0-102.0 Result Pass

-   Method II—Filter Adsorption: As filtration is an unavoidable step in    the drug product sample preparation, the filters listed in TABLE    2.11 were evaluated for the adsorption of 3,4,3-L1(1,2-HOPO):

TABLE 2.11 FILTERS EVALUATED Verification Assay Dissolution Assay 0.45μNylon Syringe Filter 10μ Dissolution Sample Inline- Filter 0.2μ PTFESyringe Filter 35μ Dissolution Sample Inline- Filter 0.2μ PVDF SyringeFilter 70μ Dissolution Sample Inline- Filter

-   -   The respective standard solution was filtered through each of        the above filter and the recovery was calculated for the        filtered and unfiltered solution. The results for the recovery        of filtered and un filtered solution were found to be within        98.0-102.0%, which indicates, there is no significant adsorption        of 3,4,3-L1(1,2-HOPO) with the above studied filters, therefore,        any of the above filters may be selected for the sample        preparation. The results of the filter study are shown below in        TABLE 2.12.

TABLE 2.12 3,4,3-LI(1,2-HOPO) - FILTER ADSORPTION EVALUATIONVerification Assay Dissolution Assay Peak % Peak % Filter Area Recovery*Filter Area Recovery* 0.45μ 3865675 100.6 10μ Filter 8669826 99.8 Nylon0.2μ PTFE 3878512 101.0 35μ Filter 8575232 98.7 0.2μ PVDF 3842269 100.070μ Filter 8637276 99.5 Un- 386492 100.6 Un- 8653392 99.6 FilteredFiltered5. Results

a. Powder in Bottle (PIB) Dosage Forms

Powder in bottle (PIB) is one of the most convenient dosage form used inearly stage clinical development because of its ease of use. Incomparison with capsules, PIB can carry large dose and fill weights.TABLE 2.13 shows the compositions of PIB evaluated. The reason forevaluating each composition is also described. The target was toidentify a suitable composition that can form uniform dispersion whendiluted with water, and also exhibit immediate drug releasecharacteristics. TABLE 2.14 describes the corresponding properties ofthe evaluated formulations.

TABLE 2.13 3,4,3-LI(1,2-HOPO) POWDER IN BOTTLE (PIB) FORMULATIONCOMPOSITIONS Formulation ID (Unit Quantity in g) A1 A2 A3 A4 A5 A6 A7 A8A9 A10 A11 Objective API API + Use of Providone as Use of Hypromellos asUse of Avicel as Ingredients ↓ → Ctrl PE dispersibility enhancerdispersibility enhancer dispersibility enhancer 3,4,3-Li(1,2-HOPO) 1.0001.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 SodiumOleate 0.092 0.092 0.092 0.092 0.092 0.092 0.092 0.092 0.092 0.092Providone, USP 0.250 0.500 1.000 (Plasdone K-29/32) Hypromellose, 0.2500.500 1.000 50 cps, USP Microcrystalline 0.250 0.500 1.000 Cellulose andCarboxymethyl Cellulose, NF (Avivel RC-591) Unite Weight (g) 1.000 1.0921.342 1.592 2.092 1.342 1.592 2.092 1.342 1.592 2.092

TABLE 2.14 3,4,3-LI(1,2-HOPO) POWDER IN BOTTLE (PIB) FORMULATIONPROPERTIES Formulation ID and Physico-Chemical Properties Parameters A1A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 Appearance Off-white color uniform blendFlow of 10 10 14 14 16 12 14 14 12 14 16 blend/ granules through Funnel(Flodex orifice dia. In mm) Bulk Density 0.51 0.53 — — — — — — — — 0.42(g/mL) Fines (% 61 63 — — — — — — — — 68 passed through mesh #40)Dispersibility Formation of sticky mass Formation Formation in 10 mL ofuniform of uniform water dispersion dispersion

The API 3,4,3-L1(1,2-HOPO) and all other compositions except A9 to A11formed a sticky mass when reconstituted in water. This behavior wasconsiderably reduced after incorporation of Avicel RC-591 (aco-processed excipient of Microcrystalline Cellulose and CarboxymethylCellulose). Composition A11 prepared using a 1:1 drug:excipient ratioformed a uniform dispersion and was therefore evaluated using the APIcontent verification assay and the dissolution in simulated gastricfluid without enzymes assay. Both assays were also performed withcomposition A2 (API+permeation enhancer sodium oleate blend) forcomparison, as described further in this report.

b. Orally Dispersible/Dissolvable Granules

Orally Dispersible/Dissolvable Granules are similar to commerciallyavailable “Sprinkles” where the granules of 3,4,3-L1(1,2-HOPO) can bedirectly transferred into the mouth from individually packedpouches/sachets and swallowed with or without water. TABLE 2.15 showsvarious compositions of orally dispersible/dissolvable granulesevaluated. The reason for evaluating each composition is also described.

TABLE 2.15 3,4,3-LI(1,2-HOPO) DISPERSIBLE/DISSOLVABLE GRANULEFORMULATION COMPOSITIONS Formulation ID (Unit Quantity in g) G1 G2 G3 G4G5 G6 G7 G8 G9 G10 G11* G12* Effect Diluents without Diluents with PVPon HPMC on Milled Milled Ingredients ↓ of → Sodium Oleate Sodium OleateGritty feel gritty feel API API Intra-Granular Materials3,4,3-Li(1,2-HOPO) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.0001.000 1.000 1.000 Sodium Oleate — — — 0.092 0.092 0.092 0.092 0.0920.092 0.092 0.092 0.092 Croscarmellose 0.075 0.075 0.075 0.075 0.0750.075 0.075 0.075 0.075 0.075 0.075 0.075 Sodium, NF (Ac-Di-Sol)Microcrystalline 1.910 — — 1.833 — — 1.821 — 1.821 — 1.833 — Celluloseand guar gum, NF (Avicel CE-15) Lactose — 1.910 — — 1.833 — — 1.821 —1.821 — 1.533 Monohydrate, NF (Pharmatose 300 M) Mannitol, USP — — 1.910— — 1.833 — — — — — — (Mannogem) Povidone, USP — — — — — — 0.012 0.012 —— — — (Plasdone K-29/32) Purified Water. USP Q.S. Q.S. Q.S. Q.S. Q.S.Q.S. Q.S. Q.S. Q.S. Q.S. Q.S. Q.S. Extra-Granular Materials Magensiumsterate, 0.015 0.015 0.015 — — — — — — — — — NF (HyQual) Hypromellose,50 cps — — — — — — — — 0.012 0.012 — 0.300 Unit Weight (g) 3.000 3.0003.000 3.000 3.000 3.000 3.000 3.000 3.000 3.000 3.000 3.000

The target was to identify a suitable composition that can impart smoothfeel in the mouth, and also exhibit immediate drug releasecharacteristics. TABLE 2.16 describes the corresponding properties ofthe evaluated formulations.

TABLE 2.16 3,4,3-LI(1,2-HOPO) DISPERSIBLE/DISSOLVABLE GRANULEFORMULATION PROPERTIES Formulation ID and Physico-Chemical PropertiesParameters G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11* G12* Flow of 10 12 12 1212 10 12 12 12 12 14 14 blend/ granules through Funnel (Flodex orificedia. In mm) Dispersibility Uniform API settled at the bottom of thebeaker Uniform Dispersion in 10 mL Dispersion water Files (% 28 32 48 2634 46 23 30 23 28 23 29 passed through mesh #40) Texture of GrittySmooth Slight granules gritty. when wetted Turns with water smooth overtime

Among various compositions evaluated, G11, which was formulated usingco-processed microcrystalline cellulose (Avicel CE-15) showed smoothfeel when wetted with water. Composition G12, which was formulated usingLactose monohydrate (Pharmatose 300 M), was also showed smooth feelafter few minutes of wetting. Based on these observations, compositionsG11 and G12 were further tested for the API verification assay and thedissolution in simulated gastric fluid without enzymes assay. Theresults are described further in this report.

c. Chewable Tablets

Chewable tablets are formulated for use in the mouth. They are usuallyuncoated, and are formulated to provide a release and absorption of theactive ingredient(s) in the mouth/buccal cavity or from stomach. TABLE2.17 shows the various chewable tablet compositions evaluated. Thetarget was to identify a suitable composition that can be formulated bydirect compression process, and shows immediate drug releasecharacteristics. TABLE 2.18 describes the corresponding properties ofthe evaluated formulations.

Compositions C13 and C21 showed satisfactory physical properties(absence of segregation, friability, and disintegration). Thesecompositions were, together with Composition C11, further tested for theAPI verification assay and the dissolution in simulated gastric fluidwithout enzymes assay. The results are described further in this report.

TABLE 2.17 3,4,3-LI(1,2-HOPO) CHEWABLE TABLET COMPOSITIONS FormulationID (Unit Quantity in g) Ingredients ↓ C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11C12 3,4,3-LI(1,2-HOPO) 1.000 1.000 1.000 1.000 1.000 0.500 0.500 1.0000.500 1.000 0.500 0.500 Sodium Oleate — — — — — — — — — — 0.046 —Croscarmellos 0.075 0.075 — 0.075 0.075 — — 0.075 0.075 0.076 0.075 —Sodium, NF (Ac-Di-Sol) Microcrystalline 1.400 — — — 0.350 — — 1.4001.900 — 1.854 — Cellulose and guar gum, FN (Avicel CE-15) Lactose —1.400 — — — — — — — — — 1.975 Monohydrate, Povidone and Crospovidone, NF(Ludipress) Lactos — — 1.475 — 1.050 1.480 0.490 — — 0.350 — —Monohydrate, NF (SuperTab 11 SD) Mannitol, USP — — — 1.400 — — — — — — —— (Mannogem) Maltodextrin, NF — — — — — — — — — — — — Magensium 0.0250.025 0.025 0.025 0.025 0.020 0.010 0.025 0.025 0.025 0.025 0.025sterate, NF (HyQual) Unit Total 2.500 2.500 2.500 2.500 2.500 2.5001.000 2.500 2.500 2.500 2.500 2.500 Weight (g) C13 C14 C15 C16 C17 C18C19 C20 C21 C11A C13A C21A 3,4,3-LI(1,2-HOPO) 0.500 0.500 0.500 0.5001.000 0.500 0.500 0.500 0.500 0.500 0.500 0.500 Sodium Oleate 0.046 — —— — — 0.046 — 0.046 0.005 0.005 0.005 Croscarmellos — 0.075 0.075 0.0750.075 0.075 0.075 0.075 0.075 0.008 — 0.008 Sodium, NF (Ac-Di-Sol)Microcrystalline — — — — — — — 0.950 0.927 0.185 — 0.093 Cellulose andguargum, FN (Avicel CE-15) Lactose 1.929 — — — — — — — — — 0.193 —Monohydrate, Povidone and Crospovidone, NF (Ludipress) Lactos — 1.900 —— — — — — — — — — Monohydrate, NF (SuperTab 11 SD) Mannitol, USP — —1.900 — — — — 0.950 0.927 — — 0.093 (Mannogem) Maltodextrin, NF — — —1.900 1.100 1.600 1.554 — — — — — Magensium 0.025 0.025 0.025 0.0250.025 0.025 0.025 0.025 0.025 0.003 0.003 0.003 sterate, NF (HyQual)Unit Total 2.500 2.500 2.500 2.500 2.200 2.200 2.200 2.500 2.500 0.2500.250 0.250 Weight (g)

TABLE 2.18 3,4,3-LI(1,2-HOPO) CHEWABLE TABLET PROPERTIES Formulation IDand Properties Parameters ↓ C1 C2 C3 C4 C5 C6 Visual — SegregationSegregation Segregation Segregation Segregation signs of segregation*Flow of Free Free Free Free Free Free blend/ flow flow flow flow flowflow granules through funnel Tablet tool 20 mm 20 mm 20 mm 20 mm 20 mm20 mm size Round Round Round Round Round Round beveled beveled beveledbeveled beveled beveled edge edge edge edge edge edge Hardness 6-7 7 116-7 8 8 (kp) Friability Fail** Fail** Fail** Fail** Fail** Fail** (%)Thickness — — — — — — (mm) Disintegration — — — — — — time (min) C13 C14C15 C16 C17 C18 Visual NA Segregation Segregation SegregationSegregation Segregation signs of segregation Flow of Free Free Free FreeFree Free blend/ flow flow flow flow flow flow granules flow throughfunnel Tablet tool 20 mm 20 mm 20 mm 20 mm 20 mm 20 mm size Round RoundRound Round Round Round beveled beveled beveled beveled beveled bevelededge edge edge edge edge edge Hardness 22 17 18 NA NA NA (kp) Friability0.57 Fail** Fail** Fail** Fail** Fail** (%) Thickness 6.23 NA NA NA NANA (mm) Disintegration 4 NA NA NA NA NA time (min) Formulation ID andProperties Parameters ↓ C7 C8 C9 C10 C11 C12 Visual Segregation NA —Segregation Segregation — signs of segregation* Flow of Free Free FreeFree Free Free blend/ flow flow flow flow flow flow granules throughfunnel Tablet tool 16 mm 20 mm 20 mm 20 mm 20 mm 20 mm size Round RoundRound Round Round Round beveled beveled beveled beveled beveled bevelededge edge edge edge edge edge Hardness 10 17 20 17 20 22 (kp) FriabilityFail** Fail** 0.35 Fail** 0.23 0.56 (%) Thickness — — NA NA 6.43 NA (mm)Disintegration — — >25 NA >25 5 time (min) (gel) C19 C20 C21 C11A^(X)C13A^(X) C21A^(X) Visual Segregation NA NA NA Segregation NA signs ofsegregation Flow of Free Free Free Free Free Free blend/ flow flow flowflow flow flow granules flow through funnel Tablet tool 20 mm 20 mm 20mm 20 mm 20 mm 20 mm size Round Round Round Round Round Round beveledbeveled beveled beveled beveled beveled edge edge edge edge edge edgeHardness NA 23 25 9 12 11 (kp) Friability Fail** 0.38 0.27 0.004 0.040.008 (%) Thickness NA NA 6.39 3.92 3.75 3.86 (mm) Disintegration NA 5 515 10 10 time (min) *3,4,3-LI(1,2-HOPO) is brown, allowing visualmonitoring of segregation after blending with excipients, Segregation isnot ideal as it may result in content uniformity issues. **>1.00% lossof tablet weight when rotated 100 times in friability tester.^(X)Smaller tablets with 50 mg API load.

d. Conventional Tablets

Various compositions and processes (Direct compression & Wetgranulation) were evaluated to formulate conventional tablets with 1000,750 and/or 500 mg drug load. The target was to identify a suitablecomposition that shows immediate drug release characteristics. Tables2.19-2.22 show various tablet compositions and processes evaluated. Thetablets were evaluated for various physical properties, and the resultsare summarized in TABLE 2.19-TABLE 2.22.

Tablet compositions T44, T45, T50, and T51 showed ideal tabletsproperties (compressibility, friability, hardness, and disintegration).Compositions T44 and T45 were prepared by wet granulation process, andCompositions 50 and 51 were prepared by direct compression. As directcompression is generally a preferred process based drug stability,manufacturing time, and cost aspects, Compositions T50 and T51 wereconsidered ideal, and their verification assay and dissolution insimulated gastric fluid without enzymes were tested. The results areshown further in this report.

TABLE 2.19 3,4,3-LI(1,2-HOPO) CONVENTIONAL TABLET COMPOSITIONS ANDPROPERTIES (COMPOSITION T1-T12) ID → T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11T12 Ingredients ↓ Process → DC DC DC DC DC DC DC DC DC DC DC DCIntra-Granular Materials 3,4,3-L1(1,2-HOPO) 1.000 1.000 1.000 1.0001.000 0.500 0.500 1.000 0.500 1.000 0.500 0.500 Sodium Oleate — — — — —— — — — — — — Croscarmellos — 0.075 — — 0.075 0.075 0.075 — 0.075 — —0.075 Sodium, NF (Ac-Di-Sol) Crosspovidone, NF — — 0.075 — — — — — —0.075 — — (Kollidone-CL M) Sodium starch — — — 0.075 — — — — — — 0.075 —glycolate, NF Microcrystalline — — — — 0.200 — — — — — — 0.200Cellulose, FN (Avicel PH 102) Lactose — — — — — 0.200 — — — — — —Monohydrate, NF (SuperTab 11 SD) Mannitol, USP — — — — — — 0.200 — — — —— (Mannogem) Magensium 0.010 0.011 0.011 0.011 — — — 0.010 0.011 0.0110.011 — sterate, NF (HyQual) Colloidal silicone — — — — — — — — — — — —dioxide, NF (Cab-O-Sil M5P) Purified Water, USP — — — — — — — — — — — —Extra-Granular Materials Croscarmellos — — — — — — — — — — — — Sodium,NF (Ac-Di-Sol) Colloidal silicone — — — — — _ — — — — — — dioxide, NF(Cab-O-Sil M5P) Magensium — — — — — — — — — — — — sterate, NF (HyQual)Unit Total 1.010 1.086 1.086 1.086 1.275 1275 1.275 1.010 1.086 1.0861.086 1.275 Weight (g) Physical Properties Flow No rat hole of blend/granules flow through Funnel Tablet Tooling/ 10 × 20 8.1 × 19.1 10 × 208.1 × 19.1 10 × 20 Dimension (mm) Hardness (kp) — — — — — — — — — — — —Friability (%) — — — — — — — — — — — — Thickness (mm) — — — — — — — — —— — — Disintegration — — — — — — — — — — — — time (min) ObservationsChipping and capping observed during compression

TABLE 2.20 3,4,3-LI(1,2-HOPO) CONVENTIONAL TABLET COMPOSITIONS ANDPROPERTIES (COMPOSITION T13-T25) ID → T13 T14 T15 T16 T17 T18 T19 T20T21 T22 T23 T24 T25 Ingredients ↓ Process → DC DC DC DC DC DC DC DC DCWG WG WG WG Intra-Granular Materials 3,4,3-LI(1,2-HOPO) 1.000 1.0001.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 SodiumOleate — — — — — — — — — — — — Croscarmellos Sodium, 0.075 0.075 — 0.075— — 0.075 0.075 0.075 0.075 0.075 0.075 0.075 NF (Ac-Di-Sol)Crosspovidone, NF — — — — 0.075 — — — — — — — (Kollidone-CL M) Sodiumstarch — — — — — 0.075 — — — — — — — glycolate, NF Microcrystalline — —— — — — 0.200 — — — 0.200 — — Cellulose, FN (Avicel PH 102) LactosMonohydrate, 0.200 — — — — — — 0.200 — — — 0.200 — NF (SuperTab 11 SD)Mannitol, USP — 0.200 — — — — — — 0.200 — — — 0.200 (Mannogem) Magensiumsterate, — — 0.010 0.011 0.011 0.011 0.013 0.013 0.013 — — — — NF(HyQual) Colloidal silicone — — 0.020 0.022 0.022 0.022 0.026 0.0260.026 — — — — dioxide, NF (Cab-O-Sil M5P) Purified Water, USP — — — — —— — — — Q.S. Q.S. Q.S. Q.S. Extra-Granular Materials CroscarmellosSodium, — — — — — — — — — — — — — NF (Ac-Di-Sol) Colloidal siliconedioxide, — — — — — — — — — — — — — NF (Cab-O-Sil M5P) Magensium sterate,— — — — — — — — — 0.011 0.013 0.013 0.013 NF (HyQual) Unit Total 1.2751.275 1.030 1108 1.108 1.108 1.314 1.314 1.314 1.086 1.288 1.288 1.288Weight (g) Physical Properties Flow No rat hole of blend/ granules flowthrough Funnel Tablet Tooling/ 10 × 20 8.1 × 19.1 10 × 20, modified ovaltooling Dimension (mm) Hardness (kp) — — — — — — — — — — — — —Friability (%) — — — — — — — — — — — — — Thickness (mm) — — — — — — — —— — — — — Disintegration — — — — — — — — — — 27 — — time (min)Observations Except for T23, all compositions showed chipping duringcompression

TABLE 2.21 3,4,3-LI(1,2-HOPO) CONVENTIONAL TABLET COMPOSITIONS ANDPROPERTIES (COMPOSITION T26-T38) Ingredients ID → T26 T27 T28 T29 T30T31 T32 T33 T34 T35 T36 T37 T38 ↓ Process → WG WG WG WG WG WG WG DC DCDC DC DC DC Intra-Granular Material 3,4,3-LI(1,2-HOPO) 1.000 1.000 1.0001.000 1.000 0.500 0.750 0.500 0.750 0.750 0.500 0.500 0.500 SodiumOleate — — — — — — — — — — — — — Croscarmellos 0.040 0.040 0.040 0.0400.040 0.040 0.040 0.075 0.075 0.075 0.050 0.050 0.040 Sodium, NF(Ac-Di-Sol) Crosspovidone, NF — — — — — — — — — — — — — (Kollidone-CL M)Sodium starch — — — — — — — — — — — — — glycolate, NF Microcrystalline0.270 0.270 0.217 — — 0.712 0.462 0.712 0.462 0.313 0.209 0.308 —Cellulose, FN (Avicel PH 102) Lactose — — — 0.217 — — — — — — — — 0.209Monohydrate, NF (SuperTab 11 SD) Mannitol, USP — — — — 0.217 — — — — — —— — (Mannogem) Magensium sterate, — — — — — — — — — — — — — NF (HyQual)Colloidal silicone — — 0.052 0.052 0.052 — — 0.013 0.013 0.012 0.0040.004 0.009 dioxide, NF (Cab-O-Sil M5P) Purified Water, USP Q.S. Q.S.Q.S. Q.S. Q.S. Q.S. Q.S. — — — — — — Extra-Granular MaterialsCroscarmellos 0.035 0.035 0.035 0.035 0.035 0.035 0.035 — — — — — —Sodium, NF (Ac-Di-Sol) Colloidal silicone — 0.026 0.026 0.026 0.026 — —— — — — — — dioxide, NF (Cab-O-Sil M5P) Magensium sterate, 0.013 0.0130.014 0.014 0.014 0.013 0.013 0.013 0.013 0.012 0.004 0.004 0.004 NF(HyQual) Unit Total 1.358 1.384 1.384 1.384 1.384 1.300 1.300 1.3131.313 1.162 0.766 0.866 0.761 Weight (g) Physical Properties Flow ofblend/ No rat hole granules flow through Funnel Tablet Tooling/ 10 × 208.1 × 19.1 Dimension (mm) Hardness (kp) 14-16 14-16 14-16 NA NA 14-1814-17 14-17 14-17 14-16 8.0-11.0 13-15 NA Friability (%) 0.18 0.19 0.18NA NA 0.19 0.22 0.21 0.20 0.16 0.14 0.16 NA Thickness (mm) 8.65 8.738.69 NA NA 8.92 8.54 8.97 8.48 6.96 5.49 5.63 NA Disintegration 26 27 26NA NA 4 10 3 8 6 9 8 NA time (min) Observations Slow disintegrationChipping Good tablets but do not contain sodium oleate Chipping

TABLE 2.22 3,4,3-LI(1,2-HOPO) CONVENTIONAL TABLET COMPOSITIONS ANDPROPERTIES (COMPOSITION T39-T51) ID T39 T40 T41 T42 T43 T44 T45 T46 T47T48 T49 T50 T51 Process Ingredients DC WG WG WG WG WG WG WG WG DC DC DCDC Intra-Granular Materials 3,4,3-LI(1,2-HOPO) 0.500 0.500 0.500 0.7501.000 0.750 0.500 0.500 0.750 0.500 0.750 0.500 0.500 Sodium Oleate — —— — 0.092 0.069 0.046 0.046 0.069 0.046 0.069 0.046 0.046 CroscarmellosSodium, NF (Ac-Di-Sol) 0.040 0.040 0.040 0.075 0.040 0.040 0.040 0.0400.040 0.075 0.075 0.084 0.092 Crosspovidone, NF (Kollidone-CL M) — — — —— — — — — — — — — Sodium starch glycolate, NF — — — — — — — — — — — — —Microcrystalline Cellulose, FN (Avicel PH 102) — — — — 0.200 0.313 0.308— — 0.308 0.313 0.410 0.501 Lactose Monohydrate, NF (SuperTab 11 SD)0.308 0.209 0.308 0.313 — — — 0.308 0.313 — — — — Mannitol, USP(Mannogem) — — — — — — — — — — — — — Magensium sterate, NF (HyQual) — —— — — — — — — — — — — Colloidal silicone dioxide, NF 0.009 0.009 0.0090.012 — — — — — 0.005 0.006 0.005 0.006 (Cab-O-Sil M5P) Purified Water,USP — — — — — 0.080 0.090 0.060 0.070 — — — — Extra-Granular MaterialsCroscarmellos Sodium NF (Ac-Di-Sol) — — — — 0.035 0.035 0.035 0.0350.035 — — — — Colloidal silicone dioxide, NF — — — — — — — — — — — — —(Cab-O-Sil MSP) Magensium sterate, NF (HyQual) 0.004 0.004 0.004 0.0060.013 0.015 0.009 0.009 0.012 0.005 0.006 0.005 0.006 Unit Total Weight(g) 0.861 0.761 0.861 1.156 1.380 1.302 1.028 0.998 1.289 0.938 1.2191.050 1.151 Physical Properties Flow of blend/granules flow throughFunnel No rat hole No rat hole, 14 mm orifice Tablet Tooling/ Dimension(mm) 8.1 × 19.1 10 × 20 8.1 × 19.1 10 × 20 9.2 × 18.9 10 × 20 9.2 × 18.9Modified oval Hardness (kp) NA 8.0-11.0 8.0-11 11.0-13.0 - 13-6 13-6 - -12.0-16.0 14-18 15-18 18-20 Friability (%) NA .014 .014 .019 - .018.015 - - .011 .009 0.12 0.13 Thickness (mm) NA 5.42 5.82 6.72 - 6.826.15 - - 5.99 7.48 7.72 7.96 Disintegration time (min) NA 11 10 14 - 1113 - - 9 11 6 4 Observations Chipping Good but no sodium Chipping Goodtablets Chipping Good (slow Good tablets oleate dissolution)

e. Selected Prototypes: Appearance and API Verification Assay

Based on the development studies performed, nine formulation prototypesthat showed immediate drug release behavior and required physicalproperties were identified and selected for further testing: Powder inbottle compositions A2 and A11, granule compositions G11 and G12,chewable tablet compositions C11, C13, and C21, and immediate releasetablet compositions T50 and T51. The appearance and packaging ofselected powder in bottle formulation prototypes A2 and A11 are shown inFIG. 7 (left) and FIG. 7 (right), respectively. The appearance andpackaging of selected granule formulation prototypes G11 and G12 areshown in FIG. 8 (left) and FIG. 8 (right), respectively. The appearanceand packaging of selected chewable tablet formulation prototypes C11,C13 and C21 are shown in FIG. 9 (left), FIG. 9 (center) and FIG. 9(right), respectively. The appearance and packaging of selected tabletformulation prototypes T50 and T51 are shown in FIG. 10 (left) and FIG.10 (right), respectively.

These selected prototype formulations were assayed for their content of3,4,3-L1(1,2-HOPO) as per the methods outlined for the contentverification assay. In general, all the tested formulations were foundto contain 90-110% of the label claim of 3,4,3-L1(1,2-HOPO). TABLE 2.23lists the verification assay values obtained for each of them.

TABLE 2.23 API CONTENT VERIFICATION ASSAY FOR SELECTED PROTOTYPEFORMULATIONS Formulation ID Prototype formulation Verification Assay (%W/W)* A2  Powder in bottle 101.2 A11 104.1 G11 Dispersible/dissolvable105.0 G12 granules 106.5 C11 Chewable tablets 104.5 C13 107.2 C21 103.0T50 Convention tablets 105.7 T51 104.6 *Values were calculated based onbulk drug substance (API) as a standard and assuming purity to be 100%

In the sample preparation from chewable tablets, the tablets were groundin a mortar and pestle due to the fact that the tablet matrix materialgels up upon exposure to the diluent thereby, hindering completeextraction into the assay medium upon either shaking (by a wrist actionshaker) or sonication. The grinding of the tablet matrix aids incomplete extraction of the analyte. Extraction from the intact dosageform in the other prototype formulations is complete as observed fromthe values in TABLE 2.23.

f. Selected Prototypes: Dissolution Assay

Gastric fluid dissolution testing of the prototype formulations wascarried out detailed in the methods section. In general, more than 80%of the API was released within 45 minutes of dissolution testing in allthe formulations tested. The results of the studies are listed in TABLE2.24-TABLE 2.27.

TABLE 2.24 GASTRIC FLUID DISSOLUTION RESULTS FOR POWER IN BOTTLEPROTOTYPES Time point(s) 10 min 15 min 30 min 45 min Inf.* FormulationID# A-2 Unit#1 96.35 99.51 100.08 101.20 100.40 Unit#2 91.21 97.08 99.4299.03 98.67 Unit#3 98.12 100.67 100.81 100.62 99.68 Average 95.2 99.1100.1 100.3 99.6 SD 3.59 1.83 0.70 1.12 0.87 RSD 3.77 1.85 0.69 1.120.87 Formulation ID# A-11 Unit#1 93.09 96.02 97.03 97.12 103.54 Unit#292.84 96.18 98.04 97.83 103.28 Unit#3 93.60 96.30 98.34 98.53 103.60Average 93.2 96.2 97.8 97.8 103.5 SD 0.39 0.14 0.69 0.71 0.17 RSD 0.410.15 0.70 0.72 0.16 *Infinity (Inf.) timepoint: 15 min at 250 rpm after45 min

TABLE 2.25 GASTRIC FLUID DISSOLUTION RESULTS FOR GRANULE PROTOTYPES Timepoint(s) 10 min 15 min 30 min 45 min Inf.* Formulation ID# G-11 Unit#1103.04 102.07 101.56 101.45 103.07 Unit#2 107.16 107.18 108.27 108.87110.31 Unit#3 106.80 106.72 106.61 107.31 107.83 Average 105.7 105.3105.5 105.9 107.1 SD 2.28 2.83 3.49 3.91 3.68 RSD 2.16 2.68 3.31 3.703.44 Formulation ID# G-12 Unit#1 79.94 90.14 101.72 102.63 103.11 Unit#281.51 90.79 103.73 104.71 105.25 Unit#3 81.44 90.19 102.27 103.58 104.20Average 81.0 90.4 102.6 103.6 104.2 SD 0.89 0.36 1.04 1.04 1.07 RSD 1.100.40 1.01 1.00 1.03 *Infinitiy (Inf.) timepoint: 15 min at 250 rpm after45 min

TABLE 2.26 GASTRIC FLUID DISSOLUTION RESULTS FOR CHEWABLE TABLETPROTOTYPES Time point(s) 10 min 15 min 30 min 45 min Inf.* FormulationID# C-11 Tablet#1 26.88 35.09 95.33 94.51 97.06 Tablet#2 13.01 21.6969.36 102.36 103.69 Tablet#3 15.69 27.93 71.94 98.38 99.91 Average 18.528.2 78.9 98.4 100.2 SD 7.36 6.71 14.31 3.93 3.33 RSD 39.71 23.75 18.143.99 3.32 Formulation ID# C-13 Tablet#1 42.55 55.99 94.99 100.92 101.97Tablet#2 42.24 62.84 101.29 104.19 104.13 Tablet#3 36.48 63.54 98.46102.78 103.25 Average 40.4 60.8 98.2 102.6 103.1 SD 3.42 4.17 3.16 1.641.09 RSD 8.45 6.86 3.21 1.60 1.05 Formulation ID# C-21 Tablet#1 42.3975.87 100.65 101.37 101.29 Tablet#2 56.86 85.14 102.60 101.68 101.83Tablet#3 52.38 81.93 98.88 100.23 99.93 Average 50.5 81.0 100.7 101.1101.0 SD 7.41 4.71 1.86 0.76 0.98 RSD 14.65 5.81 1.85 0.76 0.97*Infinitiy (Inf.) timepoint: 15 min at 250 rpm after 45 min

TABLE 2.27 GASTRIC FLUID DISSOLUTION RESULTS FOR CONVENTIONAL TABLETPROTOTYPES Time point(s) 10 min 15 min 30 min 45 min Inf.* FormulationID# T-50 Tablet#1 67.91 77.18 82.05 85.01 104.25 Tablet#2 55.87 70.1583.97 86.63 105.11 Tablet#3 50.18 62.85 76.36 80.11 015.18 Average 58.070.1 80.8 83.9 104.8 SD 9.05 7.17 3.96 3.39 0.52 RSD 15.61 10.23 4.904.05 0.49 Formulation ID# T-51 Tablet#1 91.44 92.95 94.53 96.45 103.04Tablet#2 83.54 87.30 90.26 91.48 102.25 Tablet#3 81.76 87.69 90.42 92.24104.70 Average 85.6 89.3 91.7 93.4 103.3 SD 5.15 3.16 2.42 2.68 1.25 RSD6.02 3.53 2.64 2.87 1.21 *Infinitiy (Inf.) timepoint: 15 min at 250 rpmafter 45 min

g. Selected Prototypes: Related Substance Assay

The related substances, estimated as area (%) from the chromatograms,and the chromatographic purity of 3,4,3-L1(1,2-HOPO) in selectedprototype formulations are listed in TABLE 2.28. For all testedcompositions, the amount of related substances found in the prototypeformulations is comparable to that present in the drug substance used ascontrol.

TABLE 2.28 CHROMATOGRAPHIC API PURITY AND RELATED SUBSTANCES PRESENT INPROTOTYPE FORMULATIONS Chromatographic Purity of 3,4,3-LI(1,2-HOPO) andRelated Substances Immediate Drug Release Chewable Name of SubstancePowder in Bottle Granules Tablets Tables Peak RRT Control A2 A11 G11 G12T51 C21 3,4,3-LI — 97.45 97.71 97.64 97.70 97.69 97.72 97.71 (1,2-HOPO)Unknown 0.59 0.01 0.03 0.02 0.02 0.02 0.01 0.01 Unknown 0.61 0.01 0.040.03 0.03 0.03 0.03 0.03 Unknown 0.62 0.01 0.04 0.03 0.03 0.03 0.02 0.03Unknown 0.87 0.11 0.16 0.14 0.15 0.17 0.15 0.14 Unknown 0.89 0.13 0.070.08 0.07 0.11 0.08 0.09 Unknown 1.02 0.08 0.07 0.07 0.08 0.06 0.09 0.07Unknown 1.11 0.04 0.06 0.05 0.05 0.04 0.05 0.04 Unknown 1.27 0.17 0.160.18 0.17 0.17 0.18 0.19 Unknown 1.30 0.10 0.13 0.09 0.11 0.13 0.12 0.11Unknown 1.39 0.20 0.13 0.13 0.13 0.13 0.13 0.13 Unknown 1.40 0.04 0.020.02 0.02 0.01 0.01 0.02 Unknown 1.46 0.60 0.46 0.49 0.50 0.47 0.49 0.52Unknown 1.74 0.15 0.12 0.14 0.13 0.12 0.12 0.13 Unknown 1.89 0.18 0.140.16 0.14 0.14 0.14 0.14 Unknown 2.17 0.33 0.27 0.32 0.28 0.27 0.27 0.28Unknown 2.30 0.05 0.06 0.07 0.06 0.06 0.05 0.06 Unknown 2.58 0.25 0.230.24 0.23 0.22 0.22 0.22 Unknown 2.79 0.11 0.11 0.12 0.10 0.10 0.10 0.10Unknown 0.59 0.01 0.03 0.02 0.02 0.02 0.01 0.01 Unknown 0.61 0.01 0.040.03 0.03 0.03 0.03 0.03 Unknown 0.62 0.01 0.04 0.03 0.03 0.03 0.02 0.036. Conclusion

The feasibility of developing oral formulations for 3,4,3-L1(1,2-HOPO)was evaluated. Four oral dosage forms were investigated: (i) powder inbottle, (ii) dispersible/dissolvable granules, (iii) chewable tablets,and (iv) conventional immediate release tablets. Based on the studiesperformed, nine formulation prototypes that showed immediate drugrelease behavior and required physical properties were identified andselected for API verification, gastric fluid dissolution, and relatedsubstance testing following defined liquid chromatography methods. Amongthese selected compositions, two are powder in bottle formulations, twoare granule formulations, three are chewable tablet formulations, andtwo are conventional tablet formulations. A11 assays confirmed thatthese prototypes are suitable for further development. The stability ofthese formulations will be evaluated prior to a first-in-human trial for3,4,3-L1(1,2-HOPO). These stability studies will also include capsulescontaining the powder in bottle composition A2, which may be the optimaldosage form for adjusting does levels in clinical settings.

Example 3—Evaluation of Stability of Prototype Oral Formulations of theActive Pharmaceutical Ingredient 3,4,3-L1(1,2-HOPO) Summary

The objective of this study was to evaluate the stability of prototypeoral formulations of the active pharmaceutical ingredient3,4,3-L1(1,2-HOPO) under 25° C./60% RH and 40° C./75% RH storageconditions over six months. The clinical dose of 3,4,3-L1(1,2-HOPO) isexpected to be in the range of 1-2 grams per unit. In order to retainthe flexibility of dosing lower and higher dose strengths in clinicalevaluation, several oral formulations were included in this work:

-   -   Powder for Reconstitution (500 mg)    -   Immediate Release Tablets (500 mg)    -   Chewable Tablets (500 mg)    -   Capsules (500 mg)    -   Capsules (100 mg)    -   Placebo Capsules (size 00)    -   Placebo Capsules (size 4)

The following is a summary of observations and trends observed in thestability study of 3,4,3-L1(1,2-HOPO) prototype formulations.

-   -   There was no change in physical appearance of the prototype        formulations and placebos upon storage at 25° C./60% RH and at        40° C./75% RH for 6 months.    -   The moisture content increased slightly in powder for        reconstitution prototype formulation stored at 40° C./75% RH. In        all the other formulations including the placebos the moisture        content is comparable to the values observed in TO samples.    -   The hardness of the chewable and immediate release tablets        decreased slightly upon storage when compared to their values at        TO.    -   Development of a rancid odor upon storage at 25° C./60% RH and        at 40° C./75% RH was observed in all prototype formulations        except in powder for reconstitution. The development of rancid        odor was also observed in the placebos for 3,4,3-L1(1,2-HOPO)        capsules when stored at 25° C./60% RH and at 40° C./75% RH.    -   No major changes in the dissolution profiles of the prototype        formulations were noted in the stability study samples. The        dissolution of 3,4,3-L1(1,2-HOPO) from chewable tablets and        immediate release tablets appear to be slightly faster in the        stability study samples than what was observed in TO samples.        A11 the samples tested released more than 85% of the label claim        for 3,4,3-L1(1,2-HOPO) in 45 minutes.    -   Assay, % Label claim of 3,4,3-L1(1,2-HOPO)) in prototype dosage        form was found to be between 90-110% in all the stability study        samples analyzed as well as the TO samples.    -   Chromatographic purity measured for prototype formulations in        the stability study varied slightly in the samples upon storage        at 25° C./60% RH and at 40° C./75% RH when compared to that        observed at TO.        1. Purpose of Study

The purpose of this study was to provide data that can be used tosupport research efforts. It was not conducted in accordance with U.S.Food and Drug Administration (FDA) “Good Laboratory Practice forNonclinical Laboratory Studies” (GLP) regulations, as described in 21CFR Part 58. However, the study was planned, performed, recorded, andreported in accordance with standard practices to ensure data qualityand integrity.

2. Objective of Study

The objective of this study was to evaluate the stability of prototypeoral formulations of the active pharmaceuticalingredient)3,4,3-L1(1,2-HOPO under 25° C./60% RH and 40° C./75% RHstorage conditions over six months. The clinical dose of3,4,3-L1(1,2-HOPO) is expected to be in the range of 1-2 grams per unit.In order to retain the flexibility of dosing lower and higher dosestrengths in clinical evaluation, several oral formulations wereincluded in this work:

-   -   Powder for Reconstitution (500 mg)    -   Immediate Release Tablets (500 mg)    -   Chewable Tablets (500 mg)    -   Capsules (500 mg)    -   Capsules (100 mg)    -   Placebo Capsules (size 00)    -   Placebo Capsules (size 4)        3. Experimental Design

Prototype formulations were selected based on the results of a previousformulation development study (3,4,3-L1(1,2-HOPO)—FormulationDevelopment; EXAMPLE 2), and are presented below in TABLE 3.1 and TABLE3.2.

A11 prototype formulations and placebo capsules were staged in thisstability study under 25±2° C./60±5% RH or 40±2° C./75±5% RH storageconditions for 6 months, with sampling at 1, 3, 6 months and at TO(initial). TABLE 3.3 shows the packaging configuration for all theprototype formulations.

A variety of tests were applied to each sample at each sampling timepoint, as detailed in TABLE 3.4.

TABLE 3.1 COMPOSITION OF PROTOTYPE FORMULATIONS OF 3,4,3-LI(1,2-HOPO)Quantity in unit dosage form (mg) Capsules Capsules (500 mg) (100 mg)Prototype Immediate 3,4,3-LI(1,2- 3,4,3-LI(1,2- Powder for ReleaseChewable HOPO) Blend HOPO) Blend Reconstitution Tablets Tablets Lot#FLBN- Lot #FLBN- Formulation Formulation Formulation 20131029-1-0020131029-1-4 Ingredients ID #A11 ID #T51 ID #C21 gelatin capsule gelatincapsule 3,4,3-LI-(1,2-HOPO) 500.0 500.0 500.0 500.0 100.0 Sodium Oleate46.0 46.0 46.0 55.6 11.1 Microcrystalline 500.0 — — — — Cellulose andCarboxymethyl Cellulose, NF (Avicel RC-591) Croscarmellos — 92.0 75.0 —— Sodium, NF (AC-Di- Sol) Microcrystalline — 501.0 — — — Cellulose NF(Avicel PH 102) Colloidal silicon — 6.0 — — — dioxide, (CAB-O-Sil- M5 P)Coprocessed — — 927.0 — — Microcrystalline Cellulose and Guar gum,(Avicel CE-15) Mannitol, (Mannogem — — 927.0 — — Granular 2080)Magnesium Stearate, 6.0 25.0 — — NF (HyQual) Unit Weight (mg) 1046.01151.0 2500.0 555.6 111.1

TABLE 3.2 COMPOSITION OF PLACEBO FORMULATIONS Quantity in unit dosageform (mg) Placebo for 3,4,3-LI(1,2-HOPO) Placebo for 3,4,3-LI(1,2-HOPO)Capsules, 500 mg Capsules, 100 mg Lot #FLBN-20140619-1-00 Lot#FLBN-20140619-2-4 Ingredients gelatin capsule gelatin capsule SodiumAlginate, NF 350.0 70.0 Microcrystalline 150.0 30.0 Cellulose and GuarGum (Coprocessed) Sodium Oleate 55.6 11.1 Unite Weight (mg) 555.6 111.1

TABLE 3.3 PACKAGING CONFIGURATION OF PROTOTYPE FORMULATIONS OF 3,4,3-LI(1,2-HOPO) Prototype Formulation Packaging Configuration Powder forreconstitution 1 unit in induction heat-sealed pharmaceutical gradewhite round HDPE bottles (1 oz). Closure/Cap: Polypropylene 24 mm SecRxRibbed side Text top. Immediate release 20 tablets in inductionheat-sealed white HDPE bottle (40/50 cc) with rayon coil tablets andSorb-IT desiccant canister 1 G. Closure/Cap: Polypropylene 33 mm WhiteSaf- Cap IIIA (CRC). Chewable Tablets 20 tablets in inductionheat-sealed white HDPE bottle (150 cc) with rayon coil and Sorb-ITdesiccant canister 1 G. Closure/Cap: Polypropylene 38 mm White Saf- CapIIIA (CRC). Capsules (Size 00) 20 capsules in induction heat-sealedwhite HDPE bottle (40/50 cc) with rayon coil. Closure/Cap: Polypropylene33 mm White Saf- Cap IIIA (CRC). Capsules (Size 4) 20 capsules ininduction heat-sealed white HDPE bottle (25 cc) with rayon coil.Closure/Cap: Polypropylene 28 mm White Saf- Cap IIIA (CRC). PlaceboCapsules 20 capsules in induction heat-sealed white HDPE bottle (40/50cc) with rayon (Size 00) coil. Closure/Cap: Polypropylene 33 mm WhiteSaf- Cap IIIA (CRC). Placebo Capsules 20 capsules in inductionheat-sealed white HDPE bottle (25 cc) with rayon coil. (Size 4)Closure/Cap: Polypropylene 28 mm White Saf- Cap IIIA (CRC).

TABLE 3.4 TESTS PERFORMED ON THE PROTOTYPE FORMULATIONS OF3,4,3-LI(1,2-HOPO) Physical Tests Appearance and Organoleptic MoistureChromatographic Prototypes property Content Hardness Dissolution Assaypurity Powder for X X — X X X Reconstitution Immediate X X X X X XRelease Tablets Chewable X X X X X X Tablets Capsules (00) X X — X X XCapsules (4) X X — X X X Placebo X X — — — — Capsules (00) Placebo X X —— — — Capsules (4)4. Materials and Methods

a. Test Formulation Articles and Materials

Test Article: 3,4,3-L1(1,2-HOPO)

Manufacturer: Ash Stevens, Inc. (Detroit, Mich.)

Lot Number: ML-11-276

Physical Description: Pale yellow solid

Storage Conditions: Refrigerated 2-8° C. protected from light.

Permeability Enhancer: Sodium Oleate

Manufacturer: Sigma Aldrich (St Louis, Mo.)

Lot Number: SLBH3379V

Physical Description: White powder

Storage Conditions: Refrigerated 2-8° C. protected from light.

Formulation Articles:

-   -   3,4,3-L1(1,2-HOPO) Capsules, 500 mg Lot #FLBN-20140206-2    -   3,4,3-L1(1,2-HOPO) Capsules, 100 mg Lot #FLBN-20140211-1    -   3,4,3-L1(1,2-HOPO) Powder for Reconstitution, 500 mg Lot        #FLBN-20140206-1    -   3,4,3-L1(1,2-HOPO) Immediate Release Tablets, 500 mg Lot        #FLBN-20140211-3    -   3,4,3-L1(1,2-HOPO) Chewable Tablets, 500 mg Lot #FLBN-20140211-2        Packaging Materials:    -   Bottles: 25 cc White HDPE bottle (Package All)    -   Closure/Cap: Polypropylene 28 mm White Saf-Cap IIIA (CRC)        (Package All)    -   Bottles: 40/50 cc White HDPE bottle (Package All)    -   Closure/Cap: Polypropylene 33 mm White Saf-Cap IIIA (CRC)        (Package All)    -   Bottles: 150 cc White HDPE bottle (Package All)    -   Closure/Cap: Polypropylene 38 mm White Saf-Cap IIIA (CRC)        (Package All)    -   Bottles: HDPE 1 oz Pharmaceutical Round White bottles (Drug        Plastic and Glass Company Inc)    -   Closure/Cap: Polypropylene 24 mm SecuRx Ribbed Side Text top        (Drug Plastic and Glass Company Inc)    -   Rayon Coil 12 Grain (Mfg: Carolina Absorbent Cotton)    -   Sorb-IT® 1 g, desiccant canister containing silica gel (Mfg: Sud        Chemie)        Analytical Materials:        Solvents HPLC Grade—Supplier: Fischer Scientific        Chemicals ACS Grade or equivalent        HPLC Column Agilent, Eclipse XDB-C18, 4.6×150 mm, 5 μm

b. Direct Physical Test Methods

-   Appearance: The color and appearance of the prototype dosage forms    were observed and recorded.-   Organoleptic The smell emanating from the freshly opened package of    the Property: prototype formulations was noted.-   Hardness: Hardness of the tablet samples were measured with a    calibrated hardness tester and recorded.

c. Moisture Content (by KF) Test Method

-   Sample Preparation: Moisture content of the prototype formulations    was measured using a calibrated Karl Fisher coulometric titrator.    For determination of moisture content in powder for reconstitution    samples, 2 units were emptied into a scintillation vial and the    powder sample was used for the determination as is. For    determination of moisture content in tablet samples (immediate    release and chewable), 2 tablets were crushed in a clean dry glass    pestle and mortar and the powder sample was used for analysis. For    determination of moisture content in capsule samples, 2 units were    emptied into a scintillation vial and the powder sample was used for    the determination as is.-   Analysis Procedure: An empty vial with crimp cap was weighed (W1).    About 30 mg of powder sample was accurately weighed and transferred    to the empty vial and weighed (W2). About 4 mL of methanol    (Drysolv®) was added to the sample in the vial. The gross weight of    the vial was noted (W3). The sample was agitated on a vortex mixer    for about 2 minutes and a 3 mL aliquot was withdrawn into a syringe.    The syringe was weighed (S1). The entire content of the syringe was    added to the KF titrator and the empty syringe was weighed (S2). The    weight of the sample added (S1-S2) was entered in the KF titrator.    Note: The KF Titrator was calibrated 0.1% water standard before use,    and a blank water determination was performed using methanol    (Drysolv®). Water content was calculated as:    (Water content of Sample×Weight of sample (W3−W1))−(Water content of    blank×Weight of methanol (W3−W2))=Net powder weight (W2−W1)

d. Solution Assay and Content Uniformity

-   Chromatographic Method: Assay for 3,4,3-L1(1,2-HOPO) in capsules,    powder for reconstitution, immediate release tablets, and chewable    tablet samples were performed by the following chromatographic    method.    -   Note: Due to the ability of 3,4,3-L1(1,2-HOPO) to scavenge trace        metals in acidic conditions, the HPLC system was passivated by        multiple injections of the standard solution until the % RSD of        the last five injections reached less than 2%.        Column: Waters, Symmetry C18, 2.1×150 mm, 5 μm.        Mobile Phase A: 0.1% Trifluroacetic acid in Water        Mobile Phase B: 0.1% Trifluroacetic acid in Acetonitrile        Column Temperature: 30° C.        Flow Rate: 0.5 mL/min.        Injection Volume: 10 μL        Detection: UV at 250 nm        Run Time: 12 min        Diluent (Assay): 9:1 Water: Acetonitrile        Diluent (Dissolution): Simulated Gastric Fluid without enzymes

TABLE 3.5 GRADIENT CONDITIONS Gradient Program: Time (min) A % B % 0.090 10 3.5 58 42 4.5 58 42 5.0 2 98 6.5 2 98 7.0 90 10 12.0 90 10

-   System Suitability: No Interference from the diluent/placebo    components at the retention time of 3,4,3-L1(1,2-HOPO) peak. The %    RSD for the five replicate system suitability injections is not more    than 2.0%. The check standard recovery is within 95-105%. Note:    Throughout the sample preparation and storage, the flasks were    covered with aluminum foil.-   Standard Preparation: About 25 mg of 3,4,3-L1(1,2-HOPO) was    transferred to a 50 mL volumetric flask. About 30 mL of diluent was    added to the flask, and mixed well. The volume was made up to the    mark and sonicated. The temperature of the sonication bath was kept    low with the addition of ice. Similarly, another standard was    prepared as check standard.-   Sample Preparation: 3,4,3-L1(1,2-HOPO) Powder for Reconstitution,    500 mg. Powder for reconstitution from 5 units was filled into in a    glass vial. About 104.6 mg of powder sample (equivalent to 50 mg of    3,4,3-L1(1,2-HOPO)) was accurately weighed and transferred to a    clean and dry 100 mL volumetric flask covered with aluminum foil.    About 50 mL of diluent was added, mixed well and made up to the    mark. The flasks were sonicated for 30 minutes on ice-cold water.    The samples were filtered using a 0.45μ Nylon syringe filter and the    filtrate used for assay. The samples were stored at 5° C. in the    HPLC autosampler just after preparation. The 3,4,3-L1(1,2-HOPO)    concentration in each sample solution was about 0.5 mg/mL.    -   3,4,3-L1(1,2-HOPO) Immediate Release Tablets, 500 mg. Five (5)        immediate release tablets were added to a dry 1000 mL volumetric        flask covered with aluminum foil. About 500 mL of diluent was        added, mixed well and made up to the mark. The flasks were        sonicated for 90 minutes on ice-cold water. The samples were        filtered using 0.45μ Nylon syringe filter. 2 mL of the filtrate        was diluted to 10 mL with diluent and used for assay. The        samples were stored at 5° C. in the HPLC autosampler just after        preparation. The 3,4,3-L1(1,2-HOPO) concentration in each sample        solution was about 0.5 mg/mL.    -   3,4,3-L1(1,2-HOPO) Chewable Tablets, 500 mg. Five (5) chewable        tablets were powdered with a pestle and mortar. About 250 mg of        powdered sample (50 mg equivalent of 3,4,3-L1(1,2-HOPO)) was        weighed accurately and transferred to a clean and dry 100 mL        volumetric flask covered with aluminum foil. About 50 mL of        diluent was added and mixed well. The volume was made up to 100        mL. Each sample was sonicated for about 30 min on ice-cold        water. Samples were then filtered through a 0.45μ Nylon syringe        filter and the filtrates used for assay. The samples were stored        at 5° C. in the HPLC autosampler just after preparation. The        3,4,3-L1(1,2-HOPO) concentration in each sample solution was        about 0.5 mg/mL.    -   3,4,3-L1(1,2-HOPO) Capsules, 500 mg. Five (5) capsules were        emptied in a glass vial. About 55.6 mg of powder (50 mg        equivalent of 3,4,3-L1(1,2-HOPO)) was weighed in a dry 100 mL        volumetric flask covered with aluminum foil. About 50 mL of        diluent was added, and the volume was adjusted to 100 mL after        mixing. Each sample was sonicated for about 30 min on ice-cold        water. Samples were then filtered through a 0.45μ Nylon syringe        filter and the filtrates used for assay. The samples were stored        at 5° C. in the HPLC autosampler just after preparation. The        3,4,3-L1(1,2-HOPO) concentration in each sample solution was        about 0.5 mg/mL.    -   3,4,3-L1 (1,2-HOPO) Capsules, 100 mg. Five (5) capsules were        emptied in a glass vial. About 55.6 mg of powder (50 mg        equivalent of 3,4,3-L1 (1,2-HOPO)) was weighed in a dry 100 mL        volumetric flask covered with aluminum foil. About 50 mL of        diluent was added, and the volume was adjusted to 100 mL after        mixing. Each sample was sonicated for about 30 min on ice-cold        water. Samples were then filtered through a 0.45μ Nylon syringe        filter and the filtrates used for assay. The samples were stored        at 5° C. in the HPLC autosampler just after preparation. The        3,4,3-L1(1,2-HOPO) concentration in each sample solution was        about 0.5 mg/mL.        Calculation:        Assay (% Label Claim)−AsplAstd×Wstd50×VsplSample        Wt.×DF×P100×Averag Wt.1×100LC    -   Aspl=Peak area of sample solution    -   Astd=Average peak area of 5 injections of working standard        solution    -   Wstd=Weight of 3,4,3-L1(1,2-HOPO) used as standard in mg    -   Vspl=Volume of sample solution in mL    -   DF=Dilution factor (for immediate release tablets DF=5, for        other prototypes DF=1)    -   P=Purity factor of standard material    -   LC=Label Claim (Theoretical amount of 3,4,3-L1(1,2-HOPO) per        unit dose in mg)

e. Dissolution Testing

-   Chromatographic Unit doses of 3,4,3-L1(1,2-HOPO) powder for    reconstitution,-   Method: immediate release tablets, chewable tablets, and capsules    were transferred to each dissolution vessel and dissolution testing    was performed using the following conditions:    -   Apparatus: USP Apparatus II (Paddle)    -   Temperature: 37±0.5° C.    -   Stirring Speed: 50 RPM    -   Dissolution Medium: 900 mL of Simulated Gastric Fluid (without        enzymes)    -   Medium Sampling: 5 mL at 10, 15, 30, 45 minutes and Infinity (15        minutes at 250 rpm after 45 minute time point) with media        replacement.    -   The samples were filtered through 0.45 μl nylon syringe filter        and stored at 5° C. in the HPLC autosampler just after        withdrawal.    -   Throughout the dissolution testing, vessels were covered with        aluminum foil. The capsule prototype formulations were        introduced into the dissolution vessel with the help of a        sinker.    -   The dissolution samples were analyzed by the following HPLC        method.    -   Column: Waters, Symmetry C18, 2.1×150 mm, 5 μm.    -   Mobile Phase A: 0.1% Trifluroacetic acid in Water Mobile Phase        B: 0.1% Trifluroacetic acid in Acetonitrile Column Temperature:        30° C.    -   Sample Temperature: 5° C.    -   Flow Rate: 0.5 mL/min.    -   Injection Volume: 10 μL (50 μL for 100 mg capsule dissolution        study only)    -   Detection: UV at 250 nm    -   Run Time: 12 min    -   Diluent (Assay): 9:1 Water: Acetonitrile    -   Diluent (Dissolution): Simulated Gastric Fluid without enzymes

TABLE 3.6 GRADIENT CONDITIONS Gradient Program: Time (min) A % B % 0.090 10 3.5 58 42 4.5 58 42 5.0 2 98 6.5 2 98 7.0 90 10 12.0 90 10

-   System Suitability: No Interference from the diluent/placebo    components at the retention time of 3,4,3-L1(1,2-HOPO) peak.    -   The % RSD for the five replicate system suitability injections        is not more than 2.0%.    -   The check standard recovery is within 95-105%.-   Standard Preparation: 0.1 mg/mL 3,4,3-L1(1,2-HOPO). About 25 mg of    3,4,3-L1(1,2-HOPO) was accurately weighed and transferred to a 250    mL volumetric flask. About 200 mL of diluent was added, and mixed    well. The volume of the standard was made up to the mark. The    solution was sonicated in ice-cold water till complete dissolution    of the standard. Similarly, another standard was prepared as check    standard. These set of standards were used in dissolution testing of    formulations with 100 mg dose strength.    -   0.5 mg/mL 3,4,3-L1(1,2-HOPO). About 25 mg of 3,4,3-L1(1,2-HOPO)        was accurately weighed and transferred to a 50 mL volumetric        flask. About 30 mL of diluent was added, and mixed well. The        volume of the standard was made up to the mark. The solution was        sonicated in ice-cold water till complete dissolution of the        standard. Similarly, another standard was prepared as check        standard. These set of standards were used in dissolution        testing of formulations with 500 mg dose strength.        Calculation:        % Dissolved_(n) =AsplAstd×WstdVstd×900LC×P100+5900×n=1n−n−1%        Dissolved    -   Aspl=Peak area of sample solution    -   Astd=Average peak area of five injections of working standard        solution for assay    -   Wstd=Weight of 3,4,3-L1(1,2-HOPO) used as standard in mg    -   Vstd=Volume of standard solution in mL    -   Vspl=Volume of sample solution in mL    -   DF=Dilution factor (for immediate release tablets DF=5, for        other prototypes DF=1)    -   P=Purity factor of standard material    -   LC=Label Claim (Theoretical amount of 3,4,3-L1(1,2-HOPO) per        unit dose in mg)

f. Chromatographic Purity Assessment

-   Chromatographic To determine the chromatographic purity of    3,4,3-L1(1,2-HOPO)-   Method:    -   in prototype formulations the following HPLC method was used.    -   Column: Agilent Eclipse XDB-C18,    -   4.6×150 mm, 5 μm    -   Mobile Phase A: 0.05% Formic acid in 95:5    -   Water: Acetonitrile    -   Mobile Phase B: 0.05% Formic acid in Acetonitrile    -   Column Temperature: 25° C.    -   Flow Rate: 1.0 mL/min.    -   Injection Volume: 20 μL    -   Detection: UV at 250 nm    -   Run Time: 50 min    -   Diluent (Assay): 9:1 Water: Acetonitrile    -   Diluent (Dissolution): Simulated Gastric Fluid without enzymes

TABLE 3.7 GRADIENT CONDITIONS Gradient Program: Time (min) A % B % 0.0100 0 30.0 60 40 40.0 0 100 41.0 100 0 50.0 100 0

-   System Suitability: No Interference from the diluent/placebo    components at the retention time of 3,4,3-L1(1,2-HOPO) peak.    -   The % RSD for the five replicate system suitability injections        is not more than 2.0%.    -   The check standard recovery is within 95-105%.-   Standard Stock About 50 mg of 3,4,3-L1(1,2-HOPO) was accurately    weighed and-   Solutions: transferred into a 25 mL clear volumetric flask wrapped    with aluminium foil. 20 mL of diluent was added for complete    dissolution, the standard was diluted to volume with diluent and    mixed well. Similarly, another stock was prepared as check standard    stock.-   Calibration Standards: Five mL of the standard stock solution was    pipetted to a labeled 10 mL clear volumetric flask wrapped with    aluminum foil. The stock was diluted to volume with diluent and    mixed well. The concentration of 3,4,3-L1(1,2-HOPO) in the standard    solution was about 1.0 mg/mL. Similarly a check standard solution    was prepared from the check standard stock.-   Sample Preparation: 3,4,3-L1(1,2-HOPO) Powder for Reconstitution,    500 mg. Two unit doses of powder for reconstitution prototype    formulation were transferred to a clean dry 250 mL volumetric flask.    About 200 mL of diluent was added to the sample, and shaken on a    wrist action shaker until a complete uniform dispersion was obtained    (about 40 minutes). The volume was made up to the mark with diluent    and mixed well. The sample solution was filtered using a 0.45p,    nylon syringe filter. 1 mL of the filtrate was diluted to 4 mL in a    scintillation vial with the diluent and analyzed by HPLC. The    3,4,3-L1(1,2-HOPO) concentration in the sample solution is about 1.0    mg/mL.    -   3,4,3-L1(1,2-HOPO) Immediate Release Tablets, 500 mg. Two        prototype immediate release tablets were transferred to a clean        dry 250 mL volumetric flask. About 200 mL of diluent was added        to the sample, and shaken on a wrist action shaker till a        complete uniform dispersion was obtained (about 40 minutes). The        volume was made up to the mark with diluent and mixed well. The        sample solution was filtered using a 0.45μ nylon syringe filter.        1 mL of the filtrate was diluted to 4 mL in a scintillation vial        with the diluent and analyzed by HPLC. The 3,4,3-L1(1,2-HOPO)        concentration in the sample solution is about 1.0 mg/mL.    -   3,4,3-L1(1,2-HOPO) Chewable Tablets, 500 mg. Two prototype        chewable tablets were broken into few pieces in a pestle and        mortar and was transferred to a clean dry 250 mL volumetric        flask. About 200 mL of diluent was added to the sample, and        shaken on a wrist action shaker till a complete uniform        dispersion was obtained (about 40 minutes). The volume was made        up to the mark with diluent and mixed well. The sample solution        was filtered using a 0.45μ nylon syringe filter. 1 mL of the        filtrate was diluted to 4 mL in a scintillation vial with the        diluent and analyzed by HPLC. 3,4,3-L1(1,2-HOPO) concentration        in the sample solution is about 1.0 mg/mL.    -   3,4,3-L1(1,2-HOPO) Capsules, 500 mg. Two prototype capsules were        opened and the contents added to a clean dry 250 mL volumetric        flask. About 200 mL of diluent was added to the sample, and        shaken on a wrist action shaker till a complete uniform        dispersion was obtained (about 40 minutes). The volume was made        up to the mark with diluent and mixed well. The sample solution        was filtered using a 0.45μ nylon syringe filter. 1 mL of the        filtrate was diluted to 4 mL in a scintillation vial with the        diluent and analyzed by HPLC. The 3,4,3-L1(1,2-HOPO)        concentration in the sample solution is about 1.0 mg/mL.    -   3,4,3-L1 (1,2-HOPO) Capsules, 100 mg. Two prototype capsules        were opened and the contents added to a clean dry 50 mL        volumetric flask. About 40 mL of diluent was added to the        sample, and shaken on a wrist action shaker till a complete        uniform dispersion was obtained (about 40 minutes). The volume        was made up to the mark with diluent and mixed well. The sample        solution was filtered using a 0.45μ nylon syringe filter. 1 mL        of the filtrate was diluted to 4 mL in a scintillation vial with        the diluent and analyzed by HPLC. The 3,4,3-L1(1,2-HOPO)        concentration in the sample solution is about 1.0 mg/mL.    -   Note: Throughout sample preparation and storage, the flasks were        covered with aluminum foil.-   Blank Preparation: Blank samples were prepared with placebos of the    prototype formulations using a similar preparation method as for the    samples.-   Reporting: The chromatographic purity of 3,4,3-L1(1,2-HOPO) was    reported as HPLC area (%). A11 unknown peaks from the samples with    area ≥0.03% were integrated. Peaks in sample chromatograms common to    diluent, and blank preparations were disregarded.    5. Results

Results of all tests performed on the prototype formulations aresummarized in TABLE 3.8-TABLE 3.12, with each TABLE showing the resultsfor a particular assay.

TABLE 3.8 OBSERVATIONS OF PHYSICAL APPEARANCE, MOISTURE CONTENT, ANDORGANOLEPTIC PROPERTY OF 3,4,3-LI(1,2-HOPO) PROTOTYPE FORMULATIONS UPONSTORAGE AT 25° C./60% RH AND AT 40° C./75% RH 3,4,3-LI (1,2-HOPO)Capsules, 500 mg (FLBN-20140206-2) Storage Condition 40° C./75% RH 25°C./60% RH Test T0 1 Month 3 Month 6 Month 1 Month 3 Month 6 MonthPhysical Size ‘00’ Size ‘00’ Size ‘00’ Size ‘00’ Size ‘00’ Size ‘00’Size ‘00’ Appearance capsules capsules capsules capsules capsulescapsules capsules filled with filled with filled with filled with filledwith filled with filled with off-white off-white off-white off-whiteoff-white off-white off-white colored colored colored colored coloredcolored colored powder powder powder powder powder powder powderMoisture 3.39 3.86 3.88 4.17 3.66 3.38 3.46 Content % w/w OrganolepticNone Rancid smell Rancid smell Rancid smell Rancid smell Rancid smellRancid smell property in product in product in product in product inproduct in product (smell) and bottle and bottle and bottle and bottleand bottle and bottle 3,4,3-LI (1,2-HOPO) Capsules, 100 mg(FLBN-20140211-1) Storage Condition 40° C./75% RH 25° C./60% RH Test T01 Month 3 Month 6 Month 1 Month 3 Month 6 Month Physical Size 4 Size 4Size 4 Size 4 Size 4 Size 4 Size 4 Appearance capsules capsules capsulescapsules capsules capsules capsules filled with filled with filled withfilled with filled with filled with filled with off-white off-whiteoff-white off-white off-white off-white off-white colored coloredcolored colored colored colored colored powder powder powder powderpowder powder powder Moisture 3.53 4.32 4.68 4.38 3.90 3.95 3.62 Content% w/w Organoleptic None Rancid smell Rancid smell Rancid smell Rancidsmell Rancid smell Rancid smell property in product in product inproduct in product in product in product (smell) and bottle and bottleand bottle and bottle and bottle and bottle 3,4,3-LI (1,2-HOPO) Powderfor Reconstitution, 500 mg (FLBN-20140206-1) Storage Condition 40°C./75% RH 25° C./60% RH Test T0 1 Month 3 Month 6 Month 1 Month 3 Month6 Month Physical Off-white Off-white Off-white Off-white Off-whiteOff-white Off-white Appearance colored colored colored colored coloredcolored colored powder powder powder powder powder powder powderMoisture 2.32 3.42 4.89 5.92 2.73 2.78 3.18 Content % w/w OrganolepticNone None None None None None None property (smell) 3,4,3-LI (1,2-HOPO)Chewable Tablets, 500 mg (FLBN-20140211-2) Storage Condition 40° C./75%RH 25° C./60% RH Test T0 1 Month 3 Month 6 Month 1 Month 3 Month 6 MonthPhysical Off-white Off-white Off-white Off-white Off-white Off-whiteOff-white Appearance colored, colored, colored, colored, colored,colored, colored, round flat round flat round flat round flat round flatround flat round flat bevel edged bevel edged bevel edged bevel edgedbevel edged bevel edged bevel edged uncoated uncoated uncoated uncoateduncoated uncoated uncoated tablets tablets tablets tablets tabletstablets tablets Hardness (kp) 15.4 ± 0.4* 9.1 ± 0.15 9.1 ± 0.55 8.4 ±0.06 9.7 ± 0.06 9.9 ± 0.20 9.2 ± 0.50 Moisture 2.16 2.18 1.87 1.61 2.282.00 1.81 Content % w/w Organoleptic None None Rancid smell Rancid smellNone Rancid smell Rancid smell property in product in product in productin product (smell) and bottle and bottle and bottle and bottle 3,4,3-LI(1,2-HOPO) Immediate Release Tablets, 500 mg (FLBN-20140211-3) StorageCondition 40° C./75% RH 25° C./60% RH Test T0 1 Month 3 Month 6 Month 1Month 3 Month 6 Month Physical Off-white Off-white Off-white Off-whiteOff-white Off-white Off-white Appearance colored, oval colored, ovalcolored, oval colored, oval colored, oval colored, oval colored, ovalshaped shaped shaped shaped shaped shaped shaped uncoated uncoateduncoated uncoated uncoated uncoated uncoated tablets tablets tabletstablets tablets tablets tablets Hardness (kp) 17.0 ± 0.3* 12.5 ± 0.5113.9 ± 0.42 13.8 ± 0.78 15.3 ± 0.72 13.7 ± 0.75 14.7 ± 1.82 Moisture3.55 3.73 3.89 3.9 3.76 3.35 3.80 Content % w/w Organoleptic None NoneSlight rancid Rancid smell None Slight rancid Rancid smell propertysmell in in product smell in in product (smell) product and and bottleproduct and and bottle bottle bottle

TABLE 3.9 DISSOLUTION TESTING OF 3,4,3-LI(1,2-HOPO) PROTOTYPEFORMULATIONS STORAGE AT 25°C./60% RH AND AT 40°C./75% RH 3,4,3-LI(1,2-HOPO) Capsules, 500 mg (FLBN-20140206-2) Storage Condition 40°C./75% RH 25° C./60% RH Sampling T0 1 Month 3 Month 6 Month 1 Month 3Month 6 Month Time (mins) % Dissolved % Dissolved % Dissolved %Dissolved % Dissolved % Dissolved % Dissolved 10 44.0 51.8 52.7 46.749.6 62.8 41.9 15 58.6 61.9 68.0 61.6 63.9 77.4 57.7 30 83.7 87.1 92.986.0 89.2 98.9 84.9 45 95.8 96.6 100.3 94.6 97.5 105.3 96.1 Inf 99.698.5 100.9 96.5 98.4 103.7 98.8 SD SD SD SD SD SD SD 5.5 5.3 7.1 4.6 5.25.5 7.3 5.5 3.7 6.5 4.4 5.0 6.5 5.8 4.5 3.2 4.7 3.7 3.6 5.4 5.9 3.7 2.22.2 1.0 1.1 3.0 3.9 2.8 2.3 1.7 1.8 0.6 2.7 2.8 3,4,3-LI (1,2-HOPO)Capsules, 100 mg (FLBN-20140211-1) Storage Condition 40° C./75% RH 25°C./60% RH Sampling T0 1 Month 3 Month 6 Month 1 Month 3 Month 6 MonthTime (mins) % Dissolved % Dissolved % Dissolved % Dissolved % Dissolved% Dissolved % Dissolved 10 73.0 80.8 74.9 70.6 65.2 78.4 66.7 15 87.695.8 85.9 88.0 84.8 92.4 81.9 30 101.2 104.0 98.3 97.6 95.2 102.2 101.845 100.7 105.0 98.7 99.0 95.9 100.5 102.2 Inf 100.3 104.2 98.9 97.6 96.496.8 100.4 SD SD SD SD SD SD SD 17.4 11.7 13.6 9.3 15.5 16.2 11.2 11.39.8 12.1 10.8 12.2 9.6 9.6 2.3 4.5 5.7 4.3 3.7 1.9 3.5 2.9 4.1 3.8 2.84.5 4.4 2.7 2.7 3.3 5.1 1.6 3.6 3.6 5.0 3,4,3-LI (1,2-HOPO) Powder forReconstitution, 500 mg (FLBN-20140206-1) Storage Condition 40° C./75% RH25° C./60% RH Sampling T0 1 Month 3 Month 6 Month 1 Month 3 Month 6Month Time (mins) % Dissolved % Dissolved % Dissolved % Dissolved %Dissolved % Dissolved % Dissolved 10 99.0 100.4 98.5 96.6 97.1 97.3 96.715 99.7 100.2 99.2 96.9 97.7 97.6 96.5 30 99.2 100.0 99.5 97.8 97.8 98.596.6 45 99.6 101.1 99.2 97.4 97.6 97.1 96.9 Inf 99.9 101.4 100.3 97.197.8 96.8 97.3 SD SD SD SD SD SD SD 2.7 1.2 1.4 1.9 1.1 1.6 2.4 2.8 1.12.1 1.7 0.8 1.6 2.2 2.3 1.3 1.9 2.0 1.3 1.9 2.6 2.3 1.4 1.9 1.9 1.3 1.82.4 2.6 1.3 1.6 1.9 0.9 1.8 2.2 3,4,3-LI (1,2-HOPO) Chewable Tablets,500 mg (FLBN-20140211-2) Storage Condition 40° C./75% RH 25° C./60% RHSampling T0 1 Month 3 Month 6 Month 1 Month 3 Month 6 Month Time (mins)% Dissolved % Dissolved % Dissolved % Dissolved % Dissolved % Dissolved% Dissolved 10 85.7 99.0 79.4 99.7 97.4 80.4 99.7 15 97.6 99.3 84.0101.4 100.5 89.8 101.4 30 100.3 99.3 88.6 102.2 100.4 95.1 101.8 45 98.398.9 91.2 102.1 100.4 96.3 102.1 Inf 100.9 99.3 98.6 85.7* 100.0 102.5102.1 SD SD SD SD SD SD SD 3.3 0.6 3.4 1.3 2.6 5.8 3.1 2.2 1.0 2.4 1.30.7 2.7 0.5 1.2 0.8 1.9 1.0 1.1 1.6 0.9 1.1 0.6 1.6 1.0 0.9 0.7 1.0 1.20.9 1.0 40.9 1.0 0.9 0.7 3,4,3-LI (1,2-HOPO) Immediate Release Tablets,500 mg (FLBN-20140211-3) Storage Condition 40° C./75% RH 25° C./60% RHSampling T0 1 Month 3 Month 6 Month 1 Month 3 Month 6 Month Time (mins)% Dissolved % Dissolved % Dissolved % Dissolved % Dissolved % Dissolved% Dissolved 10 33.2 50.2 49.0 39.6 52.6 41.4 39.8 15 49.3 63.3 67.3 61.866.8 60.7 61.2 30 77.0 81.0 83.7 81.5 81.3 86.5 83.8 45 86.7 85.4 89.685.2 86.6 91.3 88.8 Inf 104.9 101.0 108.6 101.5 100.6 107.1 101.8 SD SDSD SD SD SD SD 11.2 15.5 8.0 11.6 11.7 15.4 11.6 12.6 12.2 7.9 10.1 8.914.0 9.2 13.8 6.6 9.2 8.5 8.0 6.0 9.9 6.3 4.4 7.6 7.7 7.4 6.1 8.5 2.41.9 2.9 2.5 2.8 2.1 2.0 *First dissolution testing sample at infinitytimepoint did not show peak at correct RT.

TABLE 3.10 ASSAY OF 3,4,3-LI(1,2-HOPO) PROT0TYPE FORMULATIONS ST0RED AT25° C./60% RH AND AT 40° C./75% RH 3,4,3-LI (1,2-HOPO) Capsules, 500 mg(FLBN-20140206-2) Storage Condition 40° C./75% RH 25° C./60% RH Test T01 Month 3 Month 6 Month 1 Month 3 Month 6 Month Assay 103.9 103.5 99.7102.8 102.1 101.3 100.8 (% LC) 3,4,3-LI (1,2-HOPO) Capsules 100 mg(FLBN-20140211-1) Storage Condition 40° C./75% RH 25° C./60% RH Test T01 Month 3 Month 6 Month 1 Month 3 Month 6 Month Assay 101.2 100.8 95.590.5 101.0 99.4 96.9 (% LC) 3,4,3-LI (1,2-HOPO) Powder forReconstitution, 500 mg (FLBN-20140206-1) Storage Condition 40° C./75% RH25° C./60% RH Test T0 1 Month 3 Month 6 Month 1 Month 3 Month 6 MonthAssay 107.7 102.4 101.1 101.7 106.4 107.0 101.9 (% LC) 3,4,3-LI(1,2-HOPO) Capsules 500 mg (FLBN-20140211-2) Storage Condition 40°C./75% RH 25° C./60% RH Test T0 1 Month 3 Month 6 Month 1 Month 3 Month6 Month Assay 104.6 102.2 103.2 101.7 104 103.1 102.5 (% LC) 3,4,3-LI(1,2-HOPO) Capsules 500 mg (FLBN-20140211-3) Storage Condition Test 40°C./75% RH 25° C./60% RH T0 1 Month 3 Month 6 Month 1 Month 3 Month 6Month Assay 107.0 103.6 102.7 105.9 104.7 103.6 102.6 (% LC)

TABLE 3.11 CHROMATOGRAPHIC PURITY OF 3,4,3-LI(1,2-HOPO) IN PROTOTYPEFORMULATION STABILITY SAMPLES ST0RED AT 25° C./60% RH AND AT 40° C./75%RH 3,4,3-LI (1,2-HOPO) Capsules, 500 mg (FLBN-20140206-2) StorageCondition 40° C./75% RH 25° C./60% RH Test T0 1 Month 3 Month 6 Month 1Month 3 Month 6 Month Chromatographic 96.80 98.65 96.29 98.30 98.5796.17 98.30 Purity (area %) 3,4,3-LI (1,2-HOPO) Capsules, 100 mg(FLBN-20140211-1) Storage Condition 40° C./75% RH 25° C./60% RH Test T01 Month 3 Month 6 Month 1 Month 3 Month 6 Month Chromatographic 96.9498.58 96.38 98.50 98.63 96.12 98.07 Purity (area %) 3,4,3-LI (1,2-HOPO)Powder for Reconstitution, 500 mg (FLBN-20140206-1) Storage Condition40° C./75% RH 25° C./60% RH Test T0 1 Month 3 Month 6 Month 1 Month 3Month 6 Month Chromatographic 96.58 98.66 96.02 98.08 98.67 95.94 97.82Purity (area %) 3,4,3-LI (1,2-HOPO) Capsules, 500 mg (FLBN-20140211-2)Storage Condition 40° C./75% RH 25° C./60% RH Test T0 1 Month 3 Month 6Month 1 Month 3 Month 6 Month Chromatographic 96.81 98.67 96.18 98.4098.63 96.03 98.11 Purity (area %) 3,4,3-LI (1,2-HOPO) Capsules, 500 mg(FLBN-20140211-3) Storage Condition 40° C./75% RH 25° C./60% RH Test T01 Month 3 Month 6 Month 1 Month 3 Month 6 Month Chromatographic 96.7398.65 96.40 98.37 98.67 96.05 98.46 Purity (area %)

TABLE 3.12 OBSERVATIONS OF PHYSICAL APPEARANCE, MOISTURE CONTENT, ANDORGANOLEPTIC PROPERTY OF PLACEBO CAPSULES FOR 3,4,3-LI(1,2-HOPO)PROTOTYPE FORMULATIONS UPON STORAGE AT 25° C./60% RH AND AT 40° C./75%RH Storage Condition 40° C./75% RH 25° C./60% RH Test T0 1 Month 3 Month6 Month 1 Month 3 Month 6 Month Placebo for 3,4,3-LI (1,2-HOPO)Capsules, 500 mg (FLBN-20140619-1 - 00 size capsules) Physical Size ‘00’Size ‘00’ Size ‘00’ Size ‘00’ Size ‘00’ Size ‘00’ Size ‘00’ Appearancecapsules capsules capsules capsules capsules capsules capsules filledwith filled with filled with filled with filled with filled with filledwith off-shite off-white off-white off-white off-white off-whiteoff-white colored colored colored colored colored colored colored powderpowder powder powder powder powder powder Moisture 5.19 5.53 6.45 6.075.36 5.18 5.02 Content % w/w Organoleptic None Rancid Rancid RancidSlight rancid Rancid Rancid property smell in smell in smell in smell insmell in smell in (smell) product product product product productproduct and bottle and bottle and bottle and bottle and bottle andbottle Placebo for 3,4,3-LI (1,2-HOPO) Capsules, 500 mg(FLBN-20140619-2 - size 4 capsules) Physical Size 4 Size 4 Size 4 Size 4Size 4 Size 4 Size 4 Appearance capsules capsules capsules capsulescapsules capsules capsules filled with filled with filled with filledwith filled with filled with filled with off-white off-white off-whiteoff-white off-white off-white off-white colored colored colored coloredcolored colored colored powder powder powder powder powder powder powderMoisture 5.19 5.65 6.34 6.28 5.48 5.21 6.05 Content % w/w OrganolepticNone Rancid Rancid Rancid Slight rancid Rancid Rancid property smell insmell in smell in smell in smell in smell in (smell) product productproduct product product product and bottle and bottle and bottle andbottle and bottle and bottle6. Conclusion

The following is a summary of observations and trends observed in thestability study of 3,4,3-L1(1,2-HOPO) prototype formulations.

-   -   There was no change in physical appearance of the prototype        formulations and placebos upon storage at 25° C./60% RH and at        40° C./75% RH for 6 months.    -   The moisture content increased slightly in powder for        reconstitution prototype formulation stored at 40° C./75% RH. In        all the other formulations including the placebos the moisture        content is comparable to the values observed in TO samples.    -   The hardness of the chewable and immediate release tablets        decreased slightly upon storage when compared to their values at        TO.    -   Development of a rancid odor upon storage at 25° C./60% RH and        at 40° C./75% RH was observed in all prototype formulations        except in powder for reconstitution. The development of rancid        odor was also observed in the placebos for 3,4,3-L1(1,2-HOPO)        capsules when stored at 25° C./60% RH and at 40° C./75% RH.    -   No major changes in the dissolution profiles of the prototype        formulations were noted in the stability study samples. The        dissolution of 3,4,3-L1(1,2-HOPO) from chewable tablets and        immediate release tablets appear to be slightly faster in the        stability study samples than what was observed in TO samples.        A11 the samples tested released more than 85% of the label claim        for 3,4,3-L1(1,2-HOPO) in 45 minutes.    -   Assay, % Label claim of 3,4,3-L1(1,2-HOPO)) in prototype dosage        form was found to be between 90-110% in all the stability study        samples analyzed as well as the TO samples.    -   Chromatographic purity measured for prototype formulations in        the stability study varied slightly in the samples upon storage        at 25° C./60% RH and at 40° C./75% RH when compared to that        observed at TO.

Example 4—Efficacy of Repeated 3,4,3-L1(1,2-HOPO) Treatment for Removingan Intravenous Dose of ²³⁸Pu from the Body of Female and MaleSwiss-Webster Mice Summary

The objective of this study was to characterize the efficacy of3,4,3-L1(1,2-HOPO) at enhancing elimination from an internal plutoniumburden in female and male Swiss-Webster mice administered a solublecitrate complex of ²³⁸Pu and treated multiple times, starting at 24hours after the exposure. Efficacy was evaluated by comparing the tissuecontents, urinary and fecal excretions of ²³⁸Pu in treated animalsversus untreated animals.

The medical countermeasure 3,4,3-L1(1,2-HOPO) was administeredparenterally (ip) or orally (po) via consecutive injections or gavages(six daily or twelve twice daily) starting at 24 hours postcontamination at the following four selected doses: 30 μmol/kg ip, 150μmol/kg po, 300 μmol/kg po, and 600 μmol/kg po (equivalent to respectiveapproximate human doses range of 2.5, 12.5, 25, and 50 mol/kg, using theaccepted conversion system of mouse doses into human equivalentdoses—HED—based on body surface area). Oral doses also included thepermeability enhancer selected during formulation development work.

Repeated parenteral and oral treatments with 3,4,3-L1(1,2-HOPO) resultedin enhanced elimination rates and reductions in total body burden anddistinct tissue content even when the first treatment dose was delayeduntil 24 hours post-contamination. In the first cohorts euthanized atseven days, the ²³⁸Pu elimination resulting from the twice-daily dosingschemes was not as good as the corresponding once daily dosing schemewith an equivalent total daily amount of API (i.e, once-daily doses of300 and 600 mol/kg were better than twice-daily doses of 150 and 300mol/kg)), when compared with saline controls. Extending the dosingregimen from a single dose to six once-daily doses allowed for moresustained elimination rates for groups treated with 3,4,3-L1(1,2-HOPO),in comparison to saline-administered controls. At 11 dayspost-contamination, maximum decorporation efficacy was observed afterthe six once-daily parenteral doses of 3,4,3-L1(1,2-HOPO). The ²³⁸Puelimination enhancement after multiple oral treatments was stilldose-dependent, as reductions in body and tissue content were slightlygreater after 6 daily doses at 600 mol/kg than after the correspondingdosing regimen at 300 μmol/kg. Nevertheless, oral treatment with 300μmol/kg resulted in significant ²³⁸Pu full body and tissue contentreduction in comparison with saline-treated controls, with adecorporation efficacy equivalent to that of parenteral treatment withDTPA. Finally, differences were noted in excretion paths: ²³⁸Puelimination occurred predominantly through feces for3,4,3-L1(1,2-HOPO)-treated mice and through urine for DTPA-treated mice,with a lower feces to urine ²³⁸Pu ratio in females, as compared withmales.

The results of this study confirmed the efficacious dose level for oraltreatment administration: When formulated with sodium oleate and orallyadministered daily for six consecutive days, 300-600 μmol/kg dose levelsof 3,4,3-L1(1,2-HOPO) produced significant decorporation efficacy ofsoluble ²³⁸Pu in mice.

Objective of Study

The objective of this study was to characterize the efficacy of3,4,3-L1(1,2-HOPO) at enhancing elimination from an internal plutoniumburden in female and male Swiss-Webster mice administered a solublecitrate complex of ²³⁸Pu and treated multiple times, starting at 24hours after the exposure. Efficacy was evaluated by comparing the tissuecontents, urinary and fecal excretions of ²³⁸Pu in treated animalsversus untreated animals.

In this regimen-optimization study, the medical countermeasure3,4,3-L1(1,2-HOPO) was administered parenterally (ip) or orally (po) viaconsecutive injections or gavages (six daily or twelve twice daily)starting at 24 hours post contamination at the following four selecteddoses: 30 mol/kg ip, 150 μmol/kg po, 300 μmol/kg po, and 600 μmol/kg po(equivalent to respective approximate human doses range of 2.5, 12.5,25, and 50 μmol/kg, using the accepted conversion system of mouse dosesinto human equivalent doses—HED—based on body surface area). Currently,the “clinical oral dose” for this product from preliminary work rangesfrom 10 to 150 μmol/kg when administered once orally at 24 hours afterinternalization of the isotope. The selected doses correspond to thelowest doses resulting in almost maximum decorporation efficacy whenadministered once at 24 hours post-exposure. These doses had notresulted in any obvious toxicity in prior experiments.

Experimental Design

-   -   Challenge isotope: ²³⁸Pu    -   Challenge dose: 25 nCi (i.e., approximately 0.8 μCi/kg)    -   Route of contamination: Intravenous (iv) tail-vein    -   Route of treatment: Intraperitoneal Injection (ip) or Oral (po)    -   Frequency of treatment: Multiple (once- or twice-daily for 6        days) doses, starting 24 hr postcontamination    -   Treatment dose calculation: Dose calculations (mg/kg or μmol/kg)        for the decorporation agent were based on the individual body        weight measured after contamination.    -   Study Duration: 11 days, in-life

TABLE 4.1 EXPERIMENTAL DESIGN Study Design for 3,4,3-LI(1,2-HOPO)Multiple Dosing Starting at 24 Hours Post Exposure with Intravenous²³⁸Pu-Citrate in Swiss-Webster Mice.^(a) Animals Animals Culled forCulled for Animals Dose of Total Necropsy at Necropsy at Culled forTreatment Treatment Challenge Number D-7^(b) D-11^(b) Necropsy at DoseLevel Dose Agent per Randomization of (1 day- (5 day- H-1^(c) (API,Concentration Animal Group Animals recovery) recovery) (standard mice)μmol/kg (mg/mL)^(d) Treatment Time* (nCI) Control ip (6×) 9F/9M — 8F/8M1F/1M NA NA 6 daily (D 1 to D 6) 25 DTPA^(f) ip (6 × 30) 9F/9M — 8F/8M1F/1M  30  2.09 6 daily (D 1 to D 6) 25 HOPO ip (6 × 30) 9F/9M — 8F/8M1F/1M  30  3.15 6 daily (D 1 to D 6) 25 Control po (6×) 13F/13M 4F/4M8F/8M 1F/1M NA NA 6 × 2 daily (D 1 to D 6) 25 Control po (12×) 4F/4M4F/4M — — NA NA 6 daily (D 1 to D 6) 25 HOPO^(g) po (6 × 300) 13F/13M4F/4M 8F/8M 1F/1M 300 31.50 6 daily (D 1 to D 6) 25 HOPO^(g) po (12 ×150) 4F/4M 4F/4M — — 150 15.75 6 × 2 daily (D 1 to D 6) 25 HOPO^(g) po(6 × 600) 13F/13M 4F/4M 8F/8M 1F/1M 600 64.00 6 daily (D 1 to D 6) 25HOPO^(g) po (12 × 300) 4F/4M 4F/4M — — 300 31.50 6 × 2 daily (D 1 to D6) 25 ^(a)Contamination event defined as Day 0 (D-0) and treatmentdosing started on Day 1 (D-1), 24 hrs post-contamination. Contaminationachieved by intravenous injection in a warmed lateral tail vein of 0.2mL of the challenge isotope (²³⁸Pu) in 0.008M sodium citrate and 0.14Msodium chloride, pH 4. Treatments and control vehicle administered byintraperitoneal injection (ip) or oral gavage (po). ^(b)Whole animal andtissue challenge isotope content determined at two unique timepoints(D-7, D-11) post treatment administration. Excreta collected daily postcontamination until necropsy. ^(c)Day 0 culling of one animal from each5-day recovery group included to determine the mean challenge isotopeburden and baseline tissue and carcass values as % of injected dose (%ID) at 1 hour post isotope administration. ^(d)Based on a molecularweight of 750.71 g/mol for 3,4,3-LI(1,2-HOPO) (0.7507 mg/μmol) and 497.4g/mol for Ca-DTPA (04974 mg/μmol), and corresponding to a 0.25 mL dosevolume for a 35 g mouse. ^(e)Two dosing regimen investigated: size dailydoses starting at 24 h post-exposure and twelve twice-daily dosesstarting at 24 h post-exposure. Doses explored in the second armcorresponded to half of the doses explored in the first arm, to mimic afractionated dosing regimen. ^(f)Sterile solutions of Ca-DTPA andZn-DTPA in water assembled from commercial pentetic acid, calciumcarbonate, zinc oxide, and sodium hydroxide, with pH adjusted at ~7.4Ca-DTPA is given for the first dose; Zn-DTPA is administered for thesubsequent 5 doses to follow FDA recommendations. ^(g)All3,4,3-LI(1,2-HOPO) oral formulations included the permeability enhancersodium oleate (1:10, w:w), as defined by previous formulationdevelopment work.5. Materials And Methods

a. Challenge Agent

-   Challenge Agent: Pu-²³⁸-   Supplier: Eckert & Ziegler (Valencia, Calif.)-   Original Stock: Pu-²³⁸ nitrate in 4M HNO3-   Lot Number: 118521-   Injection solution: 0.008M sodium citrate, 0.14M sodium chloride, pH    4-   Activity: 0.100 μCi/mL-   Radiochemical purity (%): 99.938-   Storage Conditions: filter-sterilized, −18° C. protected from light.

b. Test and Control Articles

-   Test Article: 3,4,3-L1(1,2-HOPO)-   Manufacturer: Ash Stevens, Inc. (Detroit, Mich.)-   Lot Number: ML-11-276-   Physical Description: Pale yellow solid-   Storage Conditions: Refrigerated 2-8° C. protected from light.-   Test Article Excipient: Sodium oleate-   Manufacturer: Tokyo Chemical Industry, Inc. (Tokyo, Japan)-   Lot Number: W76EC-   Physical Description: White powder-   Storage Conditions: Room temperature (15-30° C.), protected from    light.-   Test Article Vehicle: Sterile saline for injection, USP-   Manufacturer: Baxter (Deerfield, Ill.)-   Lot Number: C880088-   Physical Description: Clear, colorless, aqueous solution-   Storage Conditions: 15-30° C. (room temperature); clear viaflex    container.-   Control Article: DTPA-   Manufacturer: Sigma-Aldrich-   Lot Number: SLBB4940V-   Physical Description: White powder-   Storage Conditions: Refrigerated 2-8° C. protected from light (amber    vial).-   Control Article: Calcium carbonate-   Manufacturer: Sigma-Aldrich-   Lot Number: MKBJ9544V-   Physical Description: White powder-   Storage Conditions: Refrigerated 2-8° C. protected from light (amber    vial).-   Control Article: Zinc oxide-   Manufacturer: Sigma-Aldrich-   Lot Number: BCBM0343V-   Physical Description: White powder-   Storage Conditions: Refrigerated 2-8° C. protected from light    (opaque plastic bottle).-   Control Article Vehicle: Sterile water-   Manufacturer: BBraun Medical Inc.-   Lot Number: J3N588-   Physical Description: Clear, colorless solution-   Storage Conditions: 15-30° C. (room temperature); clear viaflex    container.-   pH Adjustment Solution: 1N Sodium hydroxide-   Manufacturer: Sigma-Aldrich-   Lot Number: BCBH1222V-   Physical Description: Clear, colorless, aqueous solution-   Storage Conditions: Refrigerated 2-8° C. protected from light    (opaque plastic bottle).

c. Dose Formulations

-   Preparation: The test article did not come into contact with metals    (e.g., no stainless steel spatulas, syringe needles, or amber    vials). Test article and control dose formulations were prepared    within 1 day of dose administration, by weighing out the appropriate    amount of test article, dispersing in vehicle. For ip solutions, the    test article was used alone, while for oral suspensions, a mixture    of test article and excipient (90:10, w:w) was used. The pH was    adjusted to 7.0-7.4 for ip administration and 5.0-5.5 for oral    administration with sterile NaOH. Dose formulations were prepared at    room temperature and filter-sterilized.-   Storage: 2-8° C.; refrigerated; protected from light.-   Characterization: The concentration of each test article formulation    was verified by high-performance liquid chromatography-mass    spectrometry.-   Test Article Handling: Test, reference, and control article    formulations were handled with the use of eye protection, gloves,    and a protective laboratory coat.-   Correct Dose Assurance: The test article was weighed with calibrated    balances. The administration of each dose formulation was properly    documented, and the amount administered to each animal recorded.

d. Test System

Animals:

-   Species: Mouse-   Strain: Swiss-Webster-   Supplier: Simonsen Laboratories, CA-   Sex: Female and Male-   Number of Animals: 90F and 90M assigned to test (78F/78M+12F/12M    extra)-   Age at First Dose: F: 11-12 weeks; M: 11-12 weeks-   Weight at First Dose: F: 28.8±1.6 g, M: 31.5±1.3 g    -   Both female and male animals were fasted for ca. 16 hrs prior to        treatment, which explains the low average weight at first dose.-   Animal Care: General procedures for animal care and housing were in    accordance with the National Research Council (NRC) Guide for the    Care and Use of Laboratory Animals (1996) and the Animal Welfare    Standards Incorporated in 9 CFR Part 3, 1991.-   Acclimation: Mice were acclimated for three to five days before the    start of the study. The general appearance of the animals was    evaluated by the research staff.-   Housing: Individual in each cage-   Cages: Metabolism plastic (Tecniplast)-   Light Cycle: 12 hr light/12 hr dark-   Temperature: 68-77° F.-   Humidity: 30-70%-   Ventilation: 10-15 room volumes per hour, with no recirculation of    air.-   Food: Purina Certified Rodent Chow #5002 (pellets, stock cages),    Picolab Certified Rodent Meal #5053 (powder, metabolism cages), or    equivalent, ad libitum.-   Water: Water (Purified, Reverse Osmosis) was provided ad libitum.-   Bedding: Not applicable.-   Enrichment: Red plastic igloo.-   Animal Assignment:-   Day: On day of contamination.-   Randomization: Randomly assigned to treatment groups.-   Identification: Individually identified by tail ink mark.-   Welfare of the Animals: Every effort was made to minimize, if not    eliminate, pain and suffering in all animals in this study. Moribund    animals and animals experiencing undue pain and suffering were    euthanized at the discretion of the research staff

e. Experimental Procedures (In-Life Evaluations and Euthanasia)

-   Contamination: Single administration by intravenous (iv) tail-vein    injection (0.2 mL injection volume) under isoflurane anesthesia.    Three standard 0.2 mL injections were performed into polyethylene    bottles and pre-loaded with aliquots of concentrated nitric acid on    each injection day. Six female and six male mice were culled for    necropsy 1 hr post-contamination and were used as standards. A11    standards were processed and counted together with the final samples    to allow verification of the contamination dose.-   Dose Administration: Single administration by intraperitoneal    injection (ip) or oral gavage (po) under isoflurane anesthesia.-   Mortality/Morbidity: Assessed at least once daily.-   Clinical Observations: Recorded once daily (4 hr post-dose on day of    treatment), or more often as clinical signs warranted. Animals were    examined for any altered clinical signs, including gross motor and    behavioral activity, and observable changes in appearance.-   Body weights: Day 1, prior to dose administration.-   Euthanasia: Mice were sacrificed by cervical dislocation, frozen,    and dissected partially thawed to limit blood loss.-   Sample Processing: Liver, kidneys, and abdominal tissue remainder    (ATR, including intact gastrointestinal (GI) tract, reproductive    organs, spleen, urinary bladder, abdominal fat) were collected for    analysis. The skeleton was defleshed after the burning cycle by    rinsing soft tissue off with water. A11 bone samples and remaining    soft tissues were collected and digested with 6N HNO3 for analysis.    Urine and fecal pellets were collected daily from the designated    tubes in each metabolism cage.-   Method of Analysis: A11 samples were dried at 100° C., followed by    controlled high temperature burning at 575° C. The resulting ashes    were chemically treated with concentrated nitric acid. A defined    aliquot of these acidified solutions or of the urine solutions was    then transferred (at a minimum volume, to ensure a maximum    sample:cocktail ratio of 1:5) and homogenized with 1N nitric acid    and scintillation cocktail into a 20-mL scintillation vial for    liquid scintillation analysis.-   Statistical Analysis: When comparing values between groups in this    study, the term “significant” is used in the statistical sense,    indicating P<0.01 or P<0.05 by one-way analysis of variance (ANOVA)    followed by adequate post hoc analysis. The Dunnett's multiple    comparison test was used to compare the groups of animals treated    with the chelating agent to the corresponding control group that was    administered saline, while the Tukey's honestly significant    difference multiple comparison test was used to perform pair wise    comparisons between all the groups treated with the chelating agent.    Both tests were performed twice, at the 95% and 99% confidence    interval levels. A11 statistical analyses were performed using    GraphPad Prism 5 (GraphPad Software, Inc., San Diego, Calif., USA).

f. Control of Bias

While evaluating the responses of the animals and conducting theanalyses, the technical staff was aware of the treatment history of eachanimal and sample. Based on the relatively objective endpoints to beexamined, however, bias is not expected to have influenced the resultsof the study.

6. Results

The in-life portion of the study was accomplished without incident. Theaverage radiochemical recovery in the female arms was 90.6% and 88.5%for the 7-day and 11-day necropsy cohorts, respectively. The averageradiochemical recovery in the male arms was 85.7% and 86.5% for the7-day and 11-day necropsy cohorts, respectively.

No adverse effect was noted for the groups of animals treated withparenteral or oral 3,4,3-L1(1,2-HOPO) at all dose level. A11 female andmale animals in all dose groups appeared healthy and survived untiltheir respective scheduled necropsy, except for one female mouse thatwas found dead in its cage at 4 hours after its first treatment (in theonce-daily 30 μmol/kg parenteral 3,4,3-L1(1,2-HOPO) treatment group withnecropsy scheduled on day 11). A large blood clot on the left abdominalcavity wall was found during necropsy, and internal bleeding due tofailed dose administration was the probable cause of death. This mousewas not included for average dose calculations in the correspondinggroup. A few anomalies were noted during sample collection andprocessing, with no significant impact on the outcome of the study:urine samples were spilled for two female mice on a single daycollection (on day 8 for one female mouse in the 300 μmol/kg oraltreatment group with necropsy scheduled on day 11, and on day 7 for onefemale mouse in the 600 μmol/kg treatment group with necropsy scheduledon day 11).

a. Female Excretion Data Analysis

FIG. 11A shows the daily fecal elimination of ²³⁸Pu and FIG. 11B showsthe daily urinary elimination of ²³⁸Pu for all treatment groups withscheduled necropsy at 7 days post-contamination. FIG. 12A shows thedaily fecal elimination of ²³⁸Pu and FIG. 12B shows the daily urinaryelimination of ²³⁸Pu for all treatment groups with scheduled necropsy at11 days post-contamination. The cumulative urinary and fecal ²³⁸Puelimination at the different scheduled necropsy times (7 days and 11days days post-contamination, respectively) are shown graphically forall experimental groups in FIG. 11C and FIG. 12C, and numerically inTABLE 4.2A; all results are expressed as a fraction of the totalrecovered ²³⁸Pu dose.

Statistical analysis was not conducted on the daily collections, but wasconducted on the cumulative fecal, urinary, and combined excretion dataat the two scheduled necropsy times. Oral treatment with3,4,3-L1(1,2-HOPO) twice-daily at 150 and 300 μmol/kg or once-daily at300 and 600 μmol/kg resulted in significant ²³⁸Pu eliminationenhancement at 7-days postcontamination, and the groups treated with thehigher twice-daily 300 μmol/kg and once-daily 300 and 600 mol/kg3,4,3-L1(1,2-HOPO) dose levels also displayed significantly enhancedfecal excretion at the 7-day time point. At the 11-day necropsy timepoint, all treatment regimens showed significant combined excretionenhancement. However, only the 30 μmol/kg parenteral and 600 μmol/kgoral treatments resulted in significant elimination enhancement throughboth urine and feces. Daily treatment seemed to enhance urinaryexcretion compared to single treatment (see previous reports) for3,4,3-L1(1,2-HOPO).

TABLE 4.2A ²³⁸PU EXCRETION IN FEMALE MICE: AVERAGE PERCENT RECOVEREDDOSE Female 7-Day Necropsy Total Urine Total Feces Total Excretion GroupTreatment Avg (Std Dev) Avg (Std Dev) Avg (Std Dev) Saline; 2 doses PO25.25 5.14 22.64 6.27 47.89 2.26 343 (150 μmol/kg); 2 doses PO 29.027.68 33.16 7.23 62.18 2.34** 343 (300 μmol/kg); 2 doses PO 30.90 5.3635.68 6.22* 66.57 5.97** Saline; 1 dose PO 27.01 2.10 17.42 1.96 44.433.70 343 (300 μmol/kg); 1 dose PO 28.92 8.11 34.34 6.28* 63.26 3.59**343 (600 μmol/kg); 1 dose PO 36.57 2.01 30.80 5.38* 67.37 5.09** Saline;IP 21.83 5.36 28.48 6.60 50.32 4.90 DTPA (30 μmol/kg); IP 41.93 3.73**30.20 3.51 72.13 2.48** 343 (30 μmol/kg); IP 37.57 3.48** 41.80 3.66**79.37 1.92** Saline; PO 28.01 2.86 23.10 3.40 51.11 4.00 343 (300μmol/kg); PO 34.27 5.86 37.63 6.15** 71.89 2.36** 343 (600 μmol/kg); PO38.52 3.33** 34.70 2.96** 73.22 3.10** AVG (SD): *P < 0.05, **p < 0.01against corresponding saline control group

TABLE 4.2B ²³⁸PU EXCRETION IN FEMALE MICE: PERCENT INCREASE IN EXCRETIONTotal Female 7-Day Necropsy Total Urine Total Feces Excretion 343 (150μmol/kg); 2 doses PO 114.93 146.48 129.85 343 (300 μmol/kg); 2 doses PO122.37 157.58 139.02 343 (300 μmol/kg); 1 dose PO 107.09 197.06 142.38343 (600 μmol/kg); 1 dose PO 135.42 176.75 151.63 DTPA (30 μmol/kg); IP192.03 106.02 143.34 343 (30 μmol/kg); IP 172.05 146.77 157.74 343 (300μmol/kg); PO 122.35 162.86 140.66 343 (600 μmol/kg); PO 137.53 150.20143.25

FIG. 13A and FIG. 13B show the cumulative urinary, fecal, and combinedelimination of ²³⁸Pu of all treated groups relative to controls for day7 and day 11, respectively, and TABLE 4.2B summarizes the percentage ofurinary, fecal, and combined excretion enhancement over the untreatedcontrol groups. Total excretion rates were better after 6 daily oraltreatments with 600 μmol/kg 3,4,3-L1(1,2-HOPO) than after parenteralDTPA, resulting in up to 143% increase in total excretion compared tothe control groups at 11 days post-contamination. Finally fractionationof the dose seemed to reduce efficacy since twice daily doses at 150 and300 μmol/kg resulted in a lesser ²³⁸Pu elimination enhancement than thecorresponding once-daily 300 and 600 mol/kg dose levels.

b. Female Tissue Data Analysis

The full body, skeleton, and liver contents of ²³⁸Pu at the 7-day (FIG.14A) and 11-day (FIG. 14B) necropsy time points are shown graphicallyfor all experimental groups in and numerically in TABLE 4.3A; allresults are expressed as a fraction of the total recovered ²³⁸Pu dose.A11 analyzed tissues showed large reductions in tissue content of ²³⁸Pufor the groups treated with DTPA or 3,4,3-L1(1,2-HOPO), compared to thecorresponding saline control group, at all dosing regimens. A11 groupstreated with 3,4,3-L1(1,2-HOPO) showed significant reductions in fullbody content of ²³⁸Pu compared to the saline control groups and groupstreated with 3,4,3-L1(1,2-HOPO) at 300 and 600 μmol/kg once-daily showedsignificant reductions in liver, kidney, GI tract, soft tissue, andskeleton content. Finally, oral treatment with once-daily 300 μmol/kg3,4,3-L1(1,2-HOPO) resulted in a decorporation efficacy equivalent tothat of parenteral treatment with DTPA.

TABLE 4.3B shows the percentage tissue content reduction compared to thecorresponding untreated control group (for significant reductions). A11treated group displayed significant reductions, with up to 45% decreasein body burden compared to the control groups at 11 dayspostcontamination after once-daily oral treatment with 600 μmol/kg3,4,3-L1(1,2-HOPO). Data related to percent recovered dose from thefemale arm are shown in TABLE 4.6.

TABLE 4.3A ²³⁸PU RETENTION IN FEMALE MICE: AVERAGE PERCENT RECOVEREDDOSE Kidney Liver GI Tract Soft Tissue Total Bone Total Body Group (Std(Std (Std (Std (Std (Std Treatment Avg Dev) Avg Dev) Avg Dev) Avg Dev)Avg Dev) Avg Dev) Female 7-Day Necropsy Saline; 2 0.41 0.19 18.71 4.331.86 0.49 2.04 0.43 29.09 5.07 52.11 2.26 doses PO 343 (150 0.23 0.0612.83 4.08 1.20 0.16* 1.64 0.10 21.92 3.68 37.82 2.34** μmol/kg); 2doses PO 343 (300 0.18 0.09 6.85 1.80** 1.25 0.11* 1.62 0.27 23.54 4.8933.43 5.97** μmol/kg); 2 doses PO Saline; 1 0.33 0.05 16.59 4.36 1.350.22 2.32 0.10 34.97 6.96 55.57 3.70 dose PO 343 (300 0.26 0.13 10.225.60 1.20 0.17 1.74 0.25* 23.31 3.89* 36.74 3.59** μmol/kg); 1 dose PO343 (600 0.23 0.03 12.48 4.33 1.24 0.26 1.62 0.35* 17.06 1.56** 32.635.09** μmol/kg); 1 dose PO Female 11-Day Necropsy Saline; 0.26 0.0512.04 3.89 1.34 0.33 2.85 0.27 33.19 6.89 49.68 4.90 IP DTPA 0.13 0.04**4.14 1.15** 0.54 0.10** 1.66 0.33** 21.40 2.88** 27.87 2.48** (30μmol/kg); IP 343 (30 0.11 0.04** 2.79 1.31** 0.59 0.10** 1.40 0.31**15.74 2.31** 20.63 1.92** μmol/kg); IP Saline; 0.27 0.06 13.39 4.54 1.370.35 2.67 0.60 31.19 5.67 48.89 4.00 PO 343 (300 0.17 0.06* 6.02 1.71**1.02 0.21* 2.06 0.38* 18.85 2.91** 28.11 2.36** μmol/kg); 1 dose PO 343(600 0.14 0.04** 5.60 1.86** 0.83 0.22** 2.01 0.36* 18.20 2.47** 26.783.10** μmol/kg); 1 dose PO AVG (SD): *P < 0.05, **p < 0.01 againstcorresponding saline control group

TABLE 4.3B ²³⁸PU RETENTION IN FEMALE MICE: PERCENT TISSUE REDUCTION GISoft Total Total Kidney Liver Tract Tissue Bone Body Female 7-DayNecropsy 343 (150 μmol/kg); 2 43.29 31.41 35.66 19.55 24.67 27.43 dosesPO 343 (300 μmol/kg); 2 56.34 63.39 33.06 20.76 19.10 35.85 doses PO 343(300 μmol/kg); 1 20.17 38.39 11.06 25.25 33.33 33.88 doses PO 343 (600μmol/kg); 1 29.85 24.79 8.30 30.25 51.23 41.28 dose PO Female 11-DayNecropsy DTPA (30 μmol/kg); 51.24 65.62 59.99 41.56 35.51 43.90 IP 343(30 μmol/kg); IP 58.43 76.81 55.96 50.90 52.58 58.48 343 (300 μmol/kg);1 37.82 55.06 25.61 22.98 39.58 42.51 dose PO 343 (600 μmol/kg); 1 48.7758.17 39.39 24.63 41.64 45.22 dose PO

c. Male Excretion Data Analysis

FIG. 15A shows the daily fecal elimination of ²³⁸Pu and FIG. 15B showsthe daily urinary elimination of ²³⁸Pu for all treatment groups withscheduled necropsy at 7 days post-contamination. FIG. 16A shows thedaily fecal elimination of ²³⁸Pu and FIG. 16B shows the daily urinaryelimination of ²³⁸Pu for all treatment groups with scheduled necropsy at11 days post-contamination. The cumulative urinary and fecal ²³⁸Puelimination at the different scheduled necropsy times (7 days and 11days days post-contamination, respectively) are shown graphically forall experimental groups in FIG. 15C and FIG. 16C, and numerically inTABLE 4.4A; all results are expressed as a fraction of the totalrecovered ²³⁸Pu dose. Oral treatment with 3,4,3-L1(1,2-HOPO) twice-dailyat 150 and 300 μmol/kg or once-daily at 300 and 600 μmol/kg resulted insignificant ²³⁸Pu elimination enhancement and in significantly enhancedfecal excretion at 7-days post-contamination. At the 11-day necropsytime point, all treatment regimens showed significant combined excretionand fecal excretion enhancement. However, no 3,4,3-L1(1,2-HOPO) oraltreatments resulted in significant elimination enhancement through theurine.

TABLE 4.4A ²³⁸PU EXCRETION IN MALE MICE: AVERAGE PERCENT RECOVERED DOSEFemale 7-Day Total Urine Total Feces Total Excretion Necropsy (Std (Std(Std Group Treatment Avg Dev) Avg Dev) Avg Dev) Saline; 2 doses PO 18.49 5.27 17.33  2.35 35.82 3.96 343 (150 μmol/kg); 2 doses PO 23.32  1.7630.26  7.91** 53.58 6.28** 343 (300 μmol/kg); 2 doses PO 25.57  4.0534.14  1.36** 59.71 3.55** Saline; 1 dose PO 21.81  4.30 17.76  2.2439.57 5.24 343 (300 μmol/kg); 1 dose PO 24.00  3.84 40.12  0.55** 64.114.18** 343 (600 μmol/kg); 1 dose PO 25.13  4.69 40.53  3.79** 65.664.35** Saline; IP 21.95  5.69 25.53  6.65 47.48 4.63 DTPA (30 μmol/kg);IP 35.09 10.80** 35.89 10.92* 70.98 3.80** 343 (30 μmol/kg); IP 31.65 6.02* 48.24  7.04** 79.89 2.67** Saline; PO 23.98  3.74 22.36  3.1546.34 1.92 343 (300 μmol/kg); PO 25.84  3.90 40.33  4.98** 66.17 5.80*343 (600 μmol/kg); PO 27.43  8.25 44.91  8.39** 72.33 5.15** AVG (SD):*P < 0.05, **p < 0.01 against corresponding saline control group

TABLE 4.4B 238PU EXCRETION IN MALE MICE: PERCENT INCREASE IN EXCRETIONTotal Female 7-Day Necropsy Total Urine Total Feces Excretion 343 (150μmol/kg); 2 doses PO 126.15 174.63 149.61 343 (300 μmol/kg); 2 doses PO138.32 197.01 166.71 343 (300 μmol/kg); 1 dose PO 110.04 225.83 162.02343 (600 μmol/kg); 1 dose PO 115.25 228.14 165.93 DTPA (30 μmol/kg); IP159.88 140.57 149.50 343 (30 μmol/kg); IP 144.22 188.96 168.28 343 (300μmol/kg); PO 107.78 180.35 142.80 343 (600 μmol/kg); PO 114.39 200.32156.10

FIG. 17A and FIG. 17B shows the cumulative urinary, fecal, and combinedelimination of ²³⁸Pu of all treated groups relative to controls for day7 and day 11, respectively, and TABLE 4.4B summarizes the percentage ofurinary, fecal, and combined excretion enhancement over the untreatedcontrol groups. Total excretion rates were better after 6 daily oraltreatments with 600 μmol/kg 3,4,3-L1(1,2-HOPO) than after parenteralDTPA, resulting in up to 156% increase in total excretion compared tothe control groups at 11 days post-contamination. Fractionation of thedose seemed to reduce efficacy at the lower dose level since twice-dailydoses at 150 μmol/kg resulted in a lesser ²³⁸Pu elimination enhancementthan the corresponding once-daily 300 μmol/kg dose levels. However,twice-daily doses at 300 μmol/kg or once-daily 300 μmol/kg dosesresulted in equivalent ²³⁸Pu elimination enhancement. One differencethat seemed to arise during fractionation is a change in the ²³⁸Puurine:feces ratio: urine elimination was higher after fractionated doses(twice-daily regimens) than after the corresponding once-daily dosingregimen, which may indicate saturation in the hepatic clearancecapacity.

d. Male Tissue Data Analysis

The full body, skeleton, and liver contents of ²³⁸Pu at the 7-day (FIG.18A) and 11-day (FIG. 18B) necropsy time points are shown graphicallyfor all experimental groups in and numerically in TABLE 4.5A; allresults are expressed as a fraction of the total recovered ²³⁸Pu dose.A11 analyzed tissues showed large reductions in tissue content of ²³⁸Pufor the groups treated with DTPA or 3,4,3-L1(1,2-HOPO), compared to thecorresponding saline control group, at all dosing regimens. A11 groupstreated with 3,4,3-L1(1,2-HOPO) showed significant reductions in fullbody content of ²³⁸Pu compared to the saline control groups and groupstreated with 3,4,3-L1(1,2-HOPO) at 300 and 600 μmol/kg once-daily showedsignificant reductions in liver, kidney, GI tract, soft tissue, andskeleton content. Finally, oral treatment with once-daily 300 and 600μmol/kg 3,4,3-L1(1,2-HOPO) resulted in a decorporation efficacyequivalent to that of parenteral treatment with DTPA.

TABLE 4.5B shows the percentage tissue content reduction compared to thecorresponding untreated control group (for significant reductions). A11treated group displayed significant reductions, with up to 48% decreasein body burden compared to the control groups at 11 dayspostcontamination after once-daily oral treatment with 600 μmol/kg3,4,3-L1(1,2-HOPO). Data related to percent recovered dose from the malearm are shown in TABLE 4.7.

In general the decrease in tissue burdens follows similar patterns formale and female animals.

TABLE 4.5A ²³⁸PU RETENTION IN MALE MICE: AVERAGE PERCENT RECOVERED DOSEKidney Liver GI Tract Soft Tissue Total Bone Total Body Group (Std (Std(Std (Std (Std (Std Treatment Avg Dev) Avg Dev) Avg Dev) Avg Dev) AvgDev) Avg Dev) Female 7-Day Necropsy Saline; 2 0.30 0.06 26.12 6.58 1.420.24 3.00 0.23 33.34 8.74 64.18 3.96 doses PO 343 (150 0.31 0.09 21.407.47 1.67 0.35 3.16 0.33 19.87 3.20** 46.42 6.28** μmol/kg); 2 doses PO343 (300 0.18 0.03 15.12 2.09* 1.45 0.09 2.41 0.37 21.12 3.82* 40.293.55** μmol/kg); 2 doses PO Saline; 1 0.28 0.03 22.44 3.19 1.57 0.392.68 0.51 33.46 4.85 60.43 5.24 dose PO 343 (300 0.17 0.04* 12.68 2.54*1.23 0.15 1.75 0.40* 20.06 1.56** 35.89 4.18** μmol/kg); 1 dose PO343(600 0.25 0.11 12.44 4.56* 1.63 0.55 2.22 0.30 17.81 1.61* 34.344.35** μmol/kg); dose PO Female 11-Day Necropsy Saline; IP 0.33 0.1416.77 4.63 1.97 0.62 3.55 0.32 29.90 4.83 52.52 4.63 DTPA (30 0.170.05** 4.78 0.75** 0.76 0.43** 2.05 0.51** 21.26 2.83* 29.02 3.80**μmol/kg); IP 343 (30 0.12 0.03** 2.42 0.39** 0.53 0.10** 1.14 0.20**15.90 2.88* 20.11 2.67** μmol/kg); IP Saline; 0.27 0.07 18.32 3.19 1.470.38 2.84 0.75 30.13 4.73 53.66 1.92 PO 343 (300 0.15 0.03** 9.46 3.05**1.03 0.10 2.20 0.38 20.99 3.98* 33.83 5.80** μmol/kg); 1 dose PO 343(600 0.19 0.07 6.78 3.25** 0.75 0.20** 1.93 0.38** 18.02 3.37* 27.675.15** μmol/kg); 1 dose PO AVG (SD): *P < 0.05, **p < 0.01 againstcorresponding saline control group

TABLE 4.5B ²³⁸PU RETENTION IN MALE MICE: PERCENT TISSUE REDUCTION Female7-Day GI Soft Total Total Necropsy Kidney Liver Tract Tissue Bone Body343 (150 μmol/kg); 2 −4.19 18.09 −17.71 −5.31 40.39 27.68 doses PO 343(300 μmol/kg); 2 39.84 42.11 −2.24 19.73 36.64 37.23 doses PO 343 (300μmol/kg); 1 40.67 43.47 21.76 34.62 40.06 40.61 dose PO 343 (600μmol/kg); 1 11.13 44.57 −3.93 17.40 46.78 43.17 dose PO Female 11-Day GISoft Total Total Necropsy Kidney Liver Trace Tissue Bone Body DTPA (30μmol/kg); 47.89 71.51 61.32 42.09 28.92 44.74 IP 343 (30 μmol/kg); IP65.35 85.55 72.95 67.92 46.83 61.72 343 (300 μmol/kg); 1 45.60 48.3230.01 22.51 30.34 36.96 dose PO 343 (600 μmol/kg); 1 32.19 62.99 49.0632.01 40.18 48.44 dose PO7. Conclusions

Repeated parenteral and oral treatments with 3,4,3-L1(1,2-HOPO) resultedin enhanced elimination rates and reductions in total body burden anddistinct tissue content even when the first treatment dose was delayeduntil 24 hours post-contamination. In the first cohorts euthanized atseven days, the ²³⁸Pu elimination resulting from the twice-daily dosingschemes was not as good as the corresponding once daily dosing schemewith an equivalent total daily amount of API (i.e, once-daily doses of300 and 600 mol/kg were better than twice-daily doses of 150 and 300μmol/kg), when compared with saline controls. Extending the dosingregimen from a single dose to six once-daily doses allowed for moresustained elimination rates for groups treated with 3,4,3-L1(1,2-HOPO),in comparison to saline-administered controls. At 11 dayspost-contamination, maximum decorporation efficacy was observed afterthe six once-daily parenteral doses of 3,4,3-L1(1,2-HOPO). The ²³⁸Puelimination enhancement after multiple oral treatments was stilldose-dependent, as reductions in body and tissue content were slightlygreater after 6 daily doses at 600 μmol/kg than after the correspondingdosing regimen at 300 mol/kg. Nevertheless, oral treatment with 300μmol/kg resulted in significant ²³⁸Pu full body and tissue contentreduction in comparison with saline-treated controls, with adecorporation efficacy equivalent to that of parenteral treatment withDTPA. Finally, differences were noted in excretion paths: ²³⁸Puelimination occurred predominantly through feces for3,4,3-L1(1,2-HOPO)-treated mice and through urine for DTPA-treated mice,with a lower feces to urine ²³⁸Pu ratio in females, as compared withmales.

The results of this study confirmed the efficacious dose level for oraltreatment administration: When formulated with sodium oleate and orallyadministered daily for six consecutive days, 300-600 μmol/kg dose levelsof 3,4,3-L1(1,2-HOPO) produced significant decorporation efficacy ofsoluble ²³⁸Pu in mice.

a. Percent Recovered Dose for Female Arm

TABLE 4.6 PERCENT RECOVERED DOSE FEMALE ARM % Recovered Dose-FemaleAnimals-D-7 Necropsy Animal Combined Total Total Combined ID KidneyLiver ART SOFT SKEL Body Urine Feces Excreta A1 0.28% 21.55% 1.51% 1.98%28.36% 53.67% 28.69% 17.63% 46.33% A2 0.35% 12.41% 2.59% 2.61% 36.46%54.41% 24.40% 21.19% 45.59% A3 0.68% 19.26% 1.65% 2.03% 26.27% 49.89%18.32% 31.79% 50.11% A4 0.32% 21.60% 1.71% 1.56% 25.29% 50.47% 29.58%19.95% 49.53% B1 0.27%  8.04% 1.01% 1.63% 23.83% 34.79% 27.84% 37.37%65.21% B2 0.15% 11.52% 1.36% 1.69% 24.03% 38.75% 35.60% 25.66% 61.25% B30.24% 14.05% 1.12% 1.50% 23.40% 40.32% 18.65% 41.03% 59.68% B4 0.27%17.71% 1.30% 1.75% 16.40% 37.43% 33.98% 28.59% 62.57% C1 0.17%  5.50%1.12% 1.54% 28.04% 36.37% 23.02% 40.61% 63.63% C2 0.31%  9.28% 1.21%2.00% 26.98% 39.78% 33.48% 26.74% 60.22% C3 0.14%  7.15% 1.29% 1.36%21.55% 31.50% 32.25% 36.25% 68.50% C4 0.09%  5.47% 1.37% 1.57% 17.57%26.07% 34.83% 39.09% 73.93% D1 0.28% 12.55% 1.24% 2.47% 44.52% 61.05%23.99% 14.96% 38.95% D2 0.38% 19.94% 1.59% 2.27% 30.07% 54.24% 27.81%17.95% 45.76% D3 0.37% 20.77% 1.10% 2.26% 29.53% 54.04% 28.84% 17.12%45.96% D4 0.30% 13.12% 1.49% 2.28% 35.76% 52.94% 27.39% 19.67% 47.06% E10.24% 15.03% 1.20% 1.61% 18.05% 36.12% 38.49% 25.39% 63.88% E2 0.28% 5.40% 0.98% 2.12% 27.21% 35.98% 29.16% 34.86% 64.02% E3 0.43% 15.12%1.39% 1.63% 23.15% 41.72% 18.65% 39.63% 58.28% E4 0.12%  5.35% 1.24%1.58% 24.86% 33.15% 29.39% 37.46% 66.85% F1 0.21% 15.19% 1.16% 1.55%17.48% 35.58% 35.51% 28.91% 64.42% F2 0.22%  6.77% 1.24% 1.38% 15.65%25.26% 37.41% 37.33% 74.74% F3 0.22% 11.56% 0.98% 1.41% 19.07% 33.24%34.43% 32.34% 66.76% F4 0.28% 16.40% 1.59% 2.13% 16.03% 36.43% 38.95%24.62% 63.57% % Recovered Dose-Female Animals-D-11 Necropsy Animal GISoft Total Combined Total Total Combined ID Kidney Liver Tract TissueBone Body Urine Feces Excreta G1 0.25% 10.04% 1.01% 3.07% 31.33% 45.70%19.94% 34.35% 54.30% G2 0.34% 10.95% 1.11% 3.01% 34.72% 50.12% 16.92%32.96% 49.88% G3 0.23%  9.30% 1.51% 2.66% 36.12% 49.83% 29.84% 20.33%50.17% G4 0.24% 15.11% 1.07% 2.75% 36.09% 55.26% 23.37% 21.37% 44.74% G50.29%  8.39% 1.16% 2.81% 38.03% 50.69% 12.78% 36.53% 49.31% G6 0.21%14.16% 1.76% 2.33% 22.35% 40.80% 25.37% 33.83% 59.20% G7 0.31% 19.50%1.87% 3.04% 24.29% 49.01% 25.31% 25.68% 50.99% G8 0.21%  8.84% 1.26%3.12% 42.62% 56.05% 21.14% 22.81% 43.95% H1 0.10%  5.20% 0.42% 1.19%18.19% 25.11% 47.30% 27.59% 74.89% H2 0.17%  5.39% 0.69% 1.76% 17.88%25.89% 41.13% 32.98% 74.11% H3 0.11%  4.97% 0.55% 2.17% 21.46% 29.26%38.93% 31.81% 70.74% H4 0.09%  3.06% 0.41% 1.23% 20.16% 24.94% 44.88%30.18% 75.06% H5 0.10%  3.14% 0.54% 1.58% 21.81% 27.17% 39.65% 33.18%72.83% H6 0.17%  3.33% 0.60% 1.77% 25.40% 31.27% 37.66% 31.07% 68.73% H70.09%  2.77% 0.48% 1.93% 25.54% 30.80% 46.47% 22.73% 69.20% H8 0.18% 5.25% 0.62% 1.69% 20.80% 28.53% 39.42% 32.06% 71.47% I1 0.09%  4.74%0.65% 1.29% 11.21% 17.98% 43.75% 38.27% 82.02% I2 1.35% 36.77% 1.84%3.31% 32.22% 75.50% 10.74% 13.76% 24.50% I3 0.13%  4.16% 0.77% 0.99%14.80% 20.85% 37.58% 41.57% 79.15% I4 0.08%  1.79% 0.48% 1.29% 15.63%19.27% 34.92% 45.81% 80.73% I5 0.18%  1.96% 0.55% 1.94% 16.61% 21.23%39.21% 39.56% 78.77% I6 0.08%  2.48% 0.52% 1.49% 16.38% 20.95% 33.32%45.73% 79.05% I7 0.14%  3.25% 0.62% 1.62% 18.50% 24.13% 38.85% 37.01%75.87% I8 0.05%  1.16% 0.54% 1.17% 17.05% 19.99% 35.33% 44.68% 80.01% K10.24% 14.85% 1.05% 2.30% 29.96% 48.40% 30.69% 20.91% 51.60% K2 0.29%16.00% 0.95% 2.26% 35.21% 54.71% 28.99% 16.29% 45.29% K3 0.24% 12.20%1.52% 2.41% 34.03% 50.40% 24.41% 25.19% 49.60% K4 0.24% 10.83% 1.08%2.68% 37.18% 52.00% 26.06% 21.94% 48.00% K5 0.34% 15.48% 1.78% 3.89%21.14% 42.63% 32.97% 24.40% 57.37% K6 0.23% 21.50% 1.78% 1.99% 25.45%50.94% 25.32% 23.74% 49.06% K7 0.37%  9.14% 1.65% 3.06% 30.05% 44.28%28.32% 27.41% 55.72% K8 0.23%  7.11% 1.14% 2.75% 36.52% 47.75% 27.30%24.95% 52.25% L1 0.13%  8.54% 0.97% 1.66% 13.83% 25.13% 38.64% 36.23%74.87% L2 0.11%  3.78% 0.80% 1.55% 18.64% 24.87% 23.93% 51.20% 75.13% L30.25%  4.98% 1.10% 2.66% 18.99% 27.98% 41.45% 30.57% 72.02% L4 0.16% 7.97% 0.99% 2.10% 15.53% 26.74% 40.10% 33.16% 73.26% L5 0.26%  5.01%0.95% 2.26% 21.88% 30.36% 29.89% 39.74% 69.64% L6 0.14%  6.46% 1.50%1.73% 21.46% 31.29% 31.40% 37.31% 68.71% L7 0.12%  4.53% 0.96% 2.18%21.52% 29.30% 33.61% 37.09% 70.70% L8 0.20%  6.86% 0.87% 2.32% 18.92%29.18% 35.12% 35.70% 70.82% M1 0.20%  4.50% 0.80% 2.21% 19.89% 27.61%44.11% 28.28% 72.39% M2 0.14%  3.03% 0.70% 2.05% 17.79% 23.71% 38.93%37.36% 76.29% M3 0.15%  5.22% 0.91% 2.16% 12.72% 21.16% 42.60% 36.24%78.84% M4 0.10%  3.96% 0.68% 2.59% 18.57% 25.90% 37.79% 36.31% 74.10% M50.11%  5.73% 0.50% 1.56% 20.43% 28.33% 35.45% 36.22% 71.67% M6 0.14% 8.43% 1.19% 1.94% 17.82% 29.52% 37.97% 32.51% 70.48% M7 0.10%  7.92%0.81% 1.48% 20.28% 30.59% 34.47% 34.94% 69.41% M8 0.16%  6.01% 1.03%2.11% 18.12% 27.44% 36.82% 35.74% 72.56% Animal ID Day 1 Day 2 Day 3 Day4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 % Recovered Dose-FemaleAnimals-Urine per Day-Day 7 Necropsy A1 23.84% 1.24% 1.15% 0.56% 0.66%0.65% 0.59% A2 19.79% 1.29% 0.90% 0.96% 0.83% 0.45% 0.18% A3 14.67%1.14% 0.76% 0.56% 0.62% 0.39% 0.17% A4 24.46% 1.88% 1.02% 0.52% 1.06%0.38% 0.26% B1 19.78% 3.55% 1.42% 0.90% 1.07% 0.62% 0.50% B2 27.69%2.63% 1.68% 0.97% 0.75% 1.18% 0.70% B3 10.97% 3.07% 1.64% 0.97% 0.60%0.82% 0.58% B4 27.78% 1.94% 1.73% 1.01% 0.54% 0.45% 0.54% C1 13.09%3.34% 1.81% 1.57% 1.35% 1.27% 0.59% C2 23.04% 3.31% 2.36% 1.99% 1.01%1.19% 0.58% C3 23.78% 3.11% 1.62% 1.35% 1.12% 0.82% 0.46% C4 26.41%2.69% 1.73% 1.57% 0.77% 0.84% 0.83% D1 17.83% 1.80% 1.23% 1.23% 0.70%0.37% 0.82% D2 22.27% 1.98% 1.21% 1.06% 0.45% 0.43% 0.43% D3 23.54%1.69% 1.12% 1.18% 0.57% 0.34% 0.40% D4 22.78% 0.67% 0.79% 1.88% 0.78%0.32% 0.17% E1 31.95% 2.40% 1.37% 0.87% 0.59% 0.80% 0.51% E2 18.66%4.44% 1.55% 1.14% 1.04% 1.36% 0.97% E3 10.76% 2.89% 1.26% 1.30% 1.12%0.74% 0.59% E4 20.85% 3.41% 1.25% 1.61% 0.81% 0.71% 0.76% F1 29.58%2.53% 0.61% 0.61% 0.85% 0.83% 0.50% F2 29.11% 3.79% 0.83% 1.24% 1.14%0.76% 0.54% F3 26.72% 3.72% 0.55% 0.90% 1.38% 0.71% 0.45% F4 31.93%3.08% 0.48% 1.15% 1.12% 0.58% 0.62% % Recovered Dose-FemaleAnimals-Urine per Day-Day 11 Necropsy G1 16.14% 1.10% 0.52% 0.25% 0.28%0.14% 0.29% 0.21% 0.36% 0.36% 0.29% G2 11.27% 2.14% 0.55% 0.40% 0.58%0.31% 0.36% 0.10% 0.52% 0.36% 0.33% G3 23.25% 1.53% 0.90% 0.55% 0.35%0.80% 0.78% 0.41% 0.62% 0.23% 0.40% G4 19.62% 1.20% 0.40% 0.36% 0.16%0.06% 0.20% 0.23% 0.52% 0.47% 0.15% G5  8.76% 0.85% 0.37% 0.74% 0.42%0.12% 0.31% 0.39% 0.25% 0.28% 0.31% G6 21.13% 1.55% 0.82% 0.22% 0.14%0.21% 0.18% 0.25% 0.28% 0.22% 0.36% G7 20.25% 0.95% 0.44% 1.24% 0.48%0.11% 0.29% 0.38% 0.25% 0.51% 0.41% G8 15.55% 1.73% 0.80% 0.36% 0.44%0.32% 0.31% 0.28% 0.61% 0.21% 0.53% H1 25.13% 7.36% 4.29% 2.85% 2.01%1.66% 1.58% 0.73% 0.82% 0.55% 0.22% H2 23.80% 5.15% 3.95% 2.27% 1.90%1.23% 1.25% 0.39% 0.29% 0.63% 0.26% H3 11.11% 9.04% 5.05% 4.68% 2.73%2.20% 1.35% 0.78% 1.10% 0.48% 0.41% H4 25.15% 7.39% 4.23% 2.55% 1.76%1.23% 1.05% 0.48% 0.45% 0.19% 0.39% H5 19.98% 6.78% 4.17% 3.02% 1.78%1.29% 1.16% 0.48% 0.55% 0.28% 0.15% H6 19.64% 5.76% 3.53% 2.51% 2.10%1.30% 1.28% 0.50% 0.49% 0.50% 0.05% H7 25.03% 7.99% 3.93% 3.39% 2.58%1.06% 0.51% 0.53% 0.66% 0.38% 0.41% H8 19.18% 6.96% 5.61% 2.43% 1.71%1.18% 1.06% 0.28% 0.40% 0.19% 0.42% I1 33.65% 4.07% 1.90% 0.50% 0.52%0.58% 0.48% 0.36% 0.73% 0.57% 0.39% I2 10.74% I3 28.43% 2.82% 1.82%1.07% 0.53% 0.31% 0.64% 0.32% 0.82% 0.56% 0.26% I4 26.88% 2.99% 1.27%0.86% 0.75% 0.50% 0.43% 0.44% 0.15% 0.47% 0.18% I5 22.35% 9.50% 2.19%1.12% 0.97% 0.67% 0.62% 0.55% 0.49% 0.55% 0.19% I6 22.70% 4.20% 2.65%1.22% 0.46% 0.38% 0.26% 0.40% 0.15% 0.35% 0.55% I7 24.00% 5.49% 3.17%1.43% 1.23% 0.59% 0.68% 0.46% 0.50% 0.58% 0.72% I8 25.06% 4.48% 1.99%0.88% 0.75% 0.28% 0.37% 0.42% 0.42% 0.24% 0.44% K1 27.03% 1.50% 0.79%0.32% 0.22% 0.00% 0.17% 0.17% 0.37% 0.08% 0.04% K2 25.13% 1.93% 0.34%0.25% 0.26% 0.09% 0.19% 0.21% 0.25% 0.20% 0.13% K3 19.76% 1.65% 0.42%0.30% 0.16% 0.33% 0.26% 0.38% 0.67% 0.17% 0.29% K4 22.71% 1.38% 0.53%0.27% 0.25% 0.06% 0.11% 0.12% 0.13% 0.31% 0.18% K5 27.51% 2.44% 0.92%0.22% 0.48% 0.10% 0.12% 0.15% 0.15% 0.53% 0.36% K6 20.49% 1.38% 0.75%0.20% 0.14% 0.23% 0.30% 0.00% 0.24% 1.14% 0.45% K7 23.98% 1.39% 0.59%0.27% 0.26% 0.18% 0.51% 0.41% 0.13% 0.38% 0.23% K8 22.26% 1.61% 0.63%0.23% 0.47% 0.20% 0.20% 0.35% 0.57% 0.42% 0.36% L1 31.91% 3.23% 1.07%0.78% 0.37% 0.16% 0.22% 0.18% 0.45% 0.05% 0.22% L2 15.70% 2.34% 0.98%0.73% 0.50% 0.73% 0.55% 0.73% 0.90% 0.35% 0.43% L3 31.06% 3.14% 1.21%1.02% 1.17% 1.20% 0.61% 0.52% 0.65% 0.50% 0.37% L4 32.59% 3.00% 1.18%0.50% 0.53% 0.16% 0.47% 0.28% 0.27% 0.57% 0.55% L5 23.01% 2.68% 0.77%0.74% 0.59% 0.50% 0.48% 0.21% 0.38% 0.35% 0.18% L6 22.51% 3.79% 1.44%0.88% 0.64% 0.40% 0.38% 0.28% 0.40% 0.33% 0.34% L7 23.74% 4.02% 1.22%1.04% 1.13% 0.46% 0.34% 0.59% 0.26% 0.47% 0.33% L8 24.54% 5.40% 1.11%1.10% 0.86% 0.59% 0.51% 0.10% 0.43% 0.33% 0.16% M1 35.39% 3.23% 1.59%0.83% 0.71% 0.53% 0.63% 0.25% 0.27% 0.38% 0.29% M2 26.40% 5.61% 1.35%0.66% 1.31% 0.65% 1.43% 0.56% 0.22% 0.16% 0.57% M3 34.68% 3.13% 0.96%0.79% 0.38% 0.34% 0.46% 0.29% 0.67% 0.20% 0.71% M4 29.20% 2.47% 1.29%1.11% 1.04% 0.47% 0.59% 0.18% 0.56% 0.51% 0.37% M5 27.64% 3.84% 0.60%1.11% 0.49% 0.48% 0.16% 0.18% 0.38% 0.35% 0.22% M6 31.42% 2.42% 0.90%0.60% 0.60% 0.42% 0.66% 0.14% 0.35% 0.16% 0.32% M7 25.83% 2.69% 1.95%0.79% 0.54% 0.49% 0.48% 0.18% 0.56% 0.64% 0.32% M8 28.47% 3.56% 1.18%0.86% 0.83% 0.54% 0.47% 0.35% 0.05% 0.24% 0.27% *Comments: Day7-M5:urine catch found fallen over in morning/Day 8-L4: urine spilled AnimalID Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 %Recovered Dose-Female Animals-Feces per Day-Day 7 Necropsy A1  6.61% 1.46% 2.12% 1.96% 1.59% 2.11% 1.76% A2  9.23%  2.04% 2.12% 2.30% 2.41%1.94% 1.16% A3 19.65%  1.71% 2.26% 2.19% 2.22% 1.87% 1.88% A4  8.83% 2.04% 2.59% 1.51% 1.85% 1.72% 1.42% B1  8.96% 13.36% 5.58% 3.61% 2.85%1.63% 1.39% B2  4.64%  8.81% 1.92% 2.93% 2.91% 2.50% 1.95% B3 19.46% 9.40% 3.39% 2.98% 2.16% 1.90% 1.75% B4  9.41%  8.35% 2.59% 1.99% 2.39%2.12% 1.74% C1 11.51% 13.77% 4.51% 3.35% 2.71% 2.52% 2.15% C2  5.30% 8.33% 3.49% 3.00% 2.82% 2.18% 1.62% C3  8.74% 12.44% 4.66% 3.74% 2.30%2.39% 1.98% C4  7.20% 14.41% 4.88% 4.08% 2.85% 3.01% 2.66% D1  4.64% 1.49% 2.21% 2.05% 1.21% 1.90% 1.44% D2  8.84%  1.01% 1.27% 1.93% 2.28%1.37% 1.25% D3  7.52%  1.07% 1.66% 1.54% 2.24% 1.45% 1.64% D4  9.24% 0.88% 1.37% 2.37% 2.06% 1.99% 1.76% E1  6.10%  8.15% 2.89% 2.76% 1.35%1.90% 2.23% E2 10.77% 10.30% 5.25% 3.97% 1.95% 1.34% 1.29% E3 13.26% 9.61% 5.53% 4.52% 2.80% 2.01% 1.91% E4 10.08% 15.30% 2.67% 2.90% 2.17%2.18% 2.16% F1  9.32%  8.03% 2.78% 2.68% 2.30% 2.40% 1.39% F2  9.52%12.87% 4.30% 3.98% 3.01% 2.04% 1.59% F3  7.17%  9.08% 4.68% 3.14% 3.85%2.26% 2.15% F4  5.19%  6.87% 2.63% 2.89% 3.00% 1.91% 2.13% % RecoveredDose-Female Animals-Urine per Day-Day 11 Necropsy G1 15.43%  2.91% 2.82%2.69% 1.92% 1.35% 2.19% 1.59% 1.47% 0.97% 1.00% G2 12.49%  2.16% 2.98%2.70% 2.84% 2.04% 2.61% 1.90% 1.46% 1.03% 0.74% G3  7.61%  1.10% 0.68%2.34% 1.38% 1.89% 1.53% 0.74% 1.59% 0.73% 0.72% G4  7.61%  0.95% 1.65%2.47% 1.15% 1.25% 1.55% 1.18% 1.39% 1.30% 0.88% G5 14.20%  1.37% 2.71%2.98% 2.55% 3.06% 2.79% 2.87% 1.73% 1.29% 0.97% G6 12.86%  1.89% 2.84%3.34% 1.88% 2.51% 1.96% 1.53% 1.84% 1.33% 1.85% G7 12.41%  1.26% 1.53%1.08% 1.48% 1.73% 1.83% 1.03% 1.14% 1.23% 0.95% G8  4.72%  1.34% 2.86%2.48% 2.21% 3.02% 1.60% 1.02% 1.00% 1.39% 1.19% H1  9.33%  3.78% 4.12%2.95% 3.24% 1.23% 1.33% 0.66% 0.40% 0.31% 0.25% H2 10.42%  5.22% 4.15%3.74% 2.72% 2.05% 1.66% 1.22% 0.85% 0.47% 0.49% H3  5.66%  5.30% 6.39%4.22% 3.06% 2.60% 2.35% 0.77% 0.75% 0.39% 0.32% H4  9.35%  4.96% 5.55%3.34% 2.44% 1.54% 1.27% 0.75% 0.43% 0.29% 0.27% H5 11.13%  5.23% 5.41%3.22% 3.11% 1.89% 1.38% 0.78% 0.47% 0.19% 0.37% H6  9.41%  5.12% 4.29%4.38% 2.27% 1.51% 1.35% 0.99% 0.72% 0.50% 0.52% H7  6.93%  3.93% 3.92%2.99% 1.41% 0.91% 1.24% 0.49% 0.37% 0.25% 0.29% H8  9.39%  5.10% 3.16%5.19% 2.10% 1.79% 1.89% 1.24% 0.99% 0.54% 0.67% I1  7.77% 14.47% 5.15%2.76% 2.07% 1.80% 1.20% 0.79% 0.82% 0.78% 0.67% I2 13.56%  0.20% I3 9.39% 15.38% 4.91% 3.63% 2.10% 2.08% 1.23% 1.03% 0.67% 0.49% 0.65% I4 8.83% 21.84% 4.95% 3.08% 1.73% 1.82% 1.07% 0.78% 0.59% 0.52% 0.58% I5 9.40% 16.92% 3.52% 3.06% 1.67% 1.64% 1.17% 0.83% 0.61% 0.32% 0.43% I6 8.09% 19.11% 6.42% 4.72% 1.85% 2.11% 1.43% 0.67% 0.55% 0.31% 0.46% I7 8.07% 11.80% 5.63% 3.06% 1.81% 1.61% 1.94% 0.96% 0.69% 0.55% 0.89% I8 7.99% 17.77% 7.70% 4.39% 1.59% 1.48% 1.45% 0.90% 0.52% 0.45% 0.44% K1 7.86%  1.41% 1.56% 1.85% 0.96% 1.78% 1.52% 1.43% 1.26% 0.65% 0.64% K2 4.85%  1.03% 1.23% 1.02% 1.08% 0.96% 1.70% 1.15% 1.14% 0.86% 1.28% K3 8.59%  2.17% 2.08% 2.43% 1.50% 2.06% 2.28% 1.42% 0.80% 0.78% 1.08% K4 5.95%  1.71% 1.87% 1.98% 2.87% 2.32% 1.96% 1.11% 0.79% 0.69% 0.70% K5 6.07%  1.87% 2.24% 1.60% 2.26% 1.89% 2.30% 2.10% 1.37% 1.47% 1.23% K610.20%  1.42% 1.84% 1.58% 1.28% 1.11% 1.28% 1.47% 0.90% 1.09% 1.57% K7 8.73%  2.00% 3.11% 1.44% 1.82% 2.58% 2.49% 1.88% 1.13% 1.09% 1.13% K8 7.83%  2.31% 3.02% 2.05% 1.91% 2.05% 2.24% 1.29% 0.92% 0.67% 0.64% L1 5.35%  9.62% 5.07% 4.52% 2.41% 1.58% 2.68% 2.01% 0.99% 1.29% 0.70% L210.51% 11.11% 4.30% 3.28% 2.57% 1.95% 4.39% 6.27% 3.81% 1.70% 1.28% L3 3.65%  5.95% 2.41% 1.82% 6.05% 4.31% 2.50% 1.63% 0.96% 0.71% 0.58% L4 5.09%  8.55% 3.68% 3.38% 2.26% 3.12% 2.41% 1.89% 0.99% 1.04% 0.75% L5 9.71%  8.68% 5.39% 3.96% 2.77% 2.44% 2.29% 1.73% 1.05% 1.04% 0.68% L6 8.91% 12.54% 4.44% 3.67% 2.39% 1.42% 1.58% 1.02% 0.50% 0.44% 0.40% L7 9.38% 10.49% 3.21% 4.12% 2.74% 1.86% 2.14% 1.29% 0.53% 0.60% 0.72% L8 5.37%  9.01% 5.10% 5.10% 3.54% 2.87% 2.03% 1.12% 0.57% 0.41% 0.59% M1 8.44%  6.23% 2.30% 3.18% 1.90% 2.20% 1.09% 1.16% 0.89% 0.48% 0.44% M2 9.22%  7.89% 3.50% 2.79% 2.00% 3.65% 4.12% 1.96% 0.80% 0.61% 0.82% M3 6.58%  7.02% 4.87% 3.46% 3.07% 3.85% 3.24% 1.85% 0.95% 0.71% 0.63% M4 5.72% 10.02% 4.65% 4.28% 4.53% 2.48% 1.63% 1.17% 0.56% 0.70% 0.58% M5 8.33% 10.31% 3.13% 3.48% 2.38% 2.57% 2.05% 1.59% 0.83% 1.03% 0.53% M6 7.11% 12.05% 1.94% 2.77% 1.31% 1.91% 0.95% 1.23% 0.91% 1.20% 1.12% M7 8.76%  7.08% 3.83% 2.90% 2.10% 3.39% 2.29% 1.36% 1.12% 1.20% 0.91% M8 7.62%  9.14% 3.56% 3.40% 2.52% 2.63% 2.08% 1.47% 1.20% 1.18% 0.93%*Comments: Day 7-M2: few/watery feces; M7: water feces/Day 8-L2: fewfeces, M5, M7, and M8: less feces than other groups (on avg)

b. Percent Recovered Dose for Male Arm

TABLE 4.7 PERCENT RECOVERED DOSE MALE ARM % Recovered Dose-MaleAnimals-Day-7 Necropsy Animal Combined Total Total Combined ID KidneyLiver ART SOFT SKEL Body Urine Feces Excreta A1 0.34% 27.03% 1.59% 2.99%26.81% 58.77% 25.44% 15.79% 41.23% A2 0.23% 34.76% 1.26% 3.18% 24.80%642.3% 15.71% 20.06% 35.77% A3 0.36% 19.23% 1.66% 3.14% 41.18% 65.57%19.45% 14.99% 34.43% A4 0.26% 23.48% 1.17% 2.68% 40.58% 68.17% 13.35%18.48% 31.83% B1 0.29% 30.79% 2.16% 3.27% 15.55% 52.05% 25.54% 22.40%47.95% B2 0.25% 12.69% 1.57% 3.41% 20.00% 37.93% 21.58% 40.49% 62.07% B30.26% 22.32% 1.65% 2.67% 23.24% 50.13% 23.85% 26.02% 49.87% B4 0.45%19.79% 1.32% 3.29% 20.71% 45.55% 22.32% 32.13% 54.45% C1 0.22% 15.72%1.42% 2.28% 24.75% 44.39% 21.21% 34.40% 55.61% C2 0.15% 12.80% 1.55%2.12% 21.49% 38.12% 29.21% 32.67% 61.88% C3 0.21% 16.24% 1.84% 3.24%23.68% 45.21%  4.35% 50.44% 54.79% C4 0.17% 16.85% 1.39% 2.82% 17.13%38.36% 26.29% 35.35% 61.64% D1 0.29% 26.94% 1.64% 2.32% 30.14% 61.33%18.47% 20.21% 38.67% D2 0.32% 22.34% 1.65% 3.33% 39.57% 67.21% 17.95%14.84% 32.79% D3 0.25% 19.76% 1.03% 2.24% 35.09% 58.38% 24.07% 17.55%41.62% D4 0.27% 20.69% 1.96% 2.85% 29.04% 54.81% 26.74% 18.45% 45.19% E10.14%  9.72% 1.02% 1.89% 17.72% 30.49% 28.61% 40.90% 69.51% E2 0.21%15.67% 1.34% 1.96% 20.95% 40.14% 19.82% 40.04% 59.86% E3 0.13% 11.76%1.21% 1.16% 20.71% 34.97% 25.43% 39.60% 65.03% E4 0.20% 13.58% 1.33%2.00% 20.84% 37.95% 22.13% 39.91% 62.05% F1 0.17%  7.14% 1.03% 2.03%20.00% 30.37% 26.88% 42.75% 69.63% F2 0.21% 17.14% 1.92% 2.08% 18.02%39.36% 24.59% 36.05% 60.64% F3 0.42% 15.20% 2.25% 2.10% 16.63% 36.59%18.97% 44.44% 63.41% F4 0.21% 10.27% 1.33% 2.66% 16.58% 31.04% 30.09%38.86% 68.96% % Recovered Dose-Male Animals-Day-11 Necropsy Animal GISoft Total Combined Total Total Combined ID Kidney Liver Tract TissueBone Body Urine Feces Excreta G1 0.29% 18.07% 3.00%  3.47% 32.56% 57.39%10.93% 31.68% 42.61% G2 0.25% 20.65% 1.54%  3.47% 31.30% 57.21% 18.06%24.73% 42.79% G3 0.62% 23.13% 2.78%  3.91% 24.95% 55.39% 25.71% 18.91%44.61% G4 0.30% 15.89% 1.58%  3.47% 23.93% 45.17% 19.63% 35.20% 54.83%G5 0.23% 14.89% 1.44%  3.79% 26.67% 47.02% 21.67% 31.31% 52.98% G6 0.23% 8.17% 1.40%  2.87% 38.98% 51.66% 27.52% 20.82% 48.34% G7 0.46% 19.39%2.04%  3.64% 29.92% 55.46% 27.80% 16.74% 44.54% G8 0.27% 14.01% 1.94% 3.74% 30.93% 50.89% 24.29% 24.82% 49.11% H1 0.14%  5.98% 1.75%  2.40%21.31% 31.58% 42.91% 25.51% 68.42% H2 0.28%  3.55% 0.46%  1.47% 18.54%24.29% 44.82% 30.89% 74.71% H3 0.20%  5.50% 0.91%  2.34% 24.85% 33.79%42.18% 24.03% 66.21% H4 0.21%  4.81% 0.70%  2.95% 22.27% 30.93% 13.18%55.88% 69.07% H5 0.11%  4.85% 0.71%  2.10% 24.22% 32.00% 38.78% 29.23%68.00% H6 0.15%  4.26% 0.57%  1.91% 22.15% 29.03% 31.74% 39.23% 70.97%H7 0.14%  4.33% 0.52%  1.81% 16.34% 23.14% 40.70% 36.16% 76.86% H8 0.17% 4.96% 0.47%  1.44% 20.37% 27.41% 26.43% 46.15% 72.59% I1 0.08%  2.60%0.43%  1.04% 11.74% 15.90% 30.06% 54.04% 84.10% I2 0.11%  1.91% 0.47% 0.98% 17.07% 20.53% 19.00% 60.47% 79.47% I3 0.10%  2.22% 0.44%  0.83%15.87% 19.46% 28.76% 51.78% 80.54% I4 0.14%  2.29% 0.49%  1.42% 17.81%22.16% 39.36% 38.48% 77.84% I5 0.10%  3.02% 0.64%  1.37% 11.38% 16.51%34.31% 49.18% 83.49% I6 0.10%  2.09% 0.58%  1.04% 18.21% 22.02% 34.20%43.78% 77.98% I7 0.13%  2.92% 0.71%  1.22% 16.06% 21.03% 34.38% 44.59%78.97% I8 0.16%  2.34% 0.49%  1.20% 19.05% 23.25% 33.18% 43.58% 76.75%K1 0.20% 17.09% 1.22% 20.37% 12.76% 51.64% 25.42% 22.94% 48.36% K2 0.23%18.06% 1.26% 25.30% 12.61% 57.46% 21.74% 20.80% 42.54% K3 0.20% 15.87%1.21%  2.10% 33.51% 52.90% 23.59% 23.51% 47.10% K4 0.28% 22.74% 2.38% 3.06% 25.63% 54.09% 26.83% 19.07% 45.91% K5 0.37% 15.70% 1.41%  3.92%32.17% 53.57% 25.97% 20.45% 46.43% K6 0.23% 14.20% 1.39%  2.03% 36.93%54.76% 15.89% 29.35% 45.24% K7 0.31% 20.85% 1.47%  2.55% 26.21% 51.39%27.49% 21.12% 48.61% K8 0.37% 22.02% 1.39%  3.37% 26.32% 53.48% 24.87%21.65% 46.52% L1 0.10%  8.71% 0.99%  2.91% 25.77% 38.48% 19.82% 41.70%61.52% L2 0.15% 15.99% 1.04%  2.09% 24.60% 43.87% 20.73% 35.40% 56.13%L3 0.19%  9.13% 1.13%  2.06% 16.37% 28.88% 27.24% 43.87% 71.12% L4 0.19% 5.50% 1.03%  2.29% 19.43% 28.44% 24.82% 46.74% 71.56% L5 0.15%  9.51%1.21%  2.57% 22.08% 32.53% 26.43% 38.04% 64.47% L6 0.14% 10.88% 0.97% 2.02% 18.56% 32.57% 30.12% 37.30% 67.43% L7 0.14%  7.89% 0.90%  1.97%15.85% 26.76% 27.17% 46.07% 73.24% L8 0.14%  8.10% 0.93%  1.69% 25.23%36.08% 30.39% 33.53% 63.92% M1 0.08%  3.86% 0.61%  1.50% 13.99% 20.04%26.10% 53.86% 79.96% M2 0.17%  3.49% 0.50%  1.55% 15.68% 21.39% 31.92%46.68% 78.61% M3 0.28%  8.37% 0.89%  1.52% 17.58% 28.64% 24.91% 46.45%71.36% M4 0.16%  6.17% 0.48%  2.24% 25.41% 34.45% 29.67% 35.88% 65.55%M5 0.25% 12.45% 0.99%  2.53% 16.78% 33.00% 26.20% 40.81% 67.00% M6 0.24% 9.96% 1.00%  2.16% 17.24% 30.60% 9.95% 59.45% 69.40% M7 0.20%  3.83%0.73%  2.01% 18.80% 25.58% 37.71% 36.72% 74.42% M8 0.13%  6.08% 0.76% 1.95% 18.71% 27.62% 32.94% 39.43% 72.38% Animal ID Day 1 Day 2 Day 3Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 % Recovered Dose-MaleAnimals-Urine per Day-Day 7 Necropsy A1 23.45% 0.94% 0.58% 0.00% 0.27%0.00% 0.20% A2 13.24% 0.92% 0.45% 0.45% 0.37% 0.17% 0.11% A3 17.58%0.33% 0.66% 0.37% 0.18% 0.19% 0.13% A4  9.90% 1.60% 0.89% 0.23% 0.24%0.13% 0.35% B1 20.65% 2.02% 0.61% 1.30% 0.61% 0.18% 0.17% B2 15.62%2.81% 1.15% 0.41% 0.58% 0.64% 0.37% B3 19.53% 2.18% 1.12% 0.54% 0.10%0.10% 0.28% B4 17.05% 1.96% 1.18% 0.89% 0.65% 0.10% 0.50% C1 12.15%4.28% 1.32% 1.17% 0.65% 0.70% 0.94% C2 23.06% 3.19% 0.98% 0.68% 0.33%0.52% 0.45% C3  2.56% 0.38% 0.50% 0.55% 0.19% 0.07% 0.11% C4 22.38%1.72% 0.95% 0.59% 0.35% 0.19% 0.10% D1 15.56% 1.09% 0.86% 0.32% 0.34%0.21% 0.10% D2 13.36% 2.69% 0.85% 0.34% 0.62% 0.09% 0.00% D3 22.21%0.84% 0.44% 0.18% 0.08% 0.19% 0.14% D4 24.80% 0.68% 0.42% 0.24% 0.13%0.23% 0.23% E1 21.72% 3.64% 1.49% 0.67% 0.39% 0.49% 0.21% E2 14.01%2.67% 0.90% 0.69% 0.62% 0.53% 0.41% E3 18.66% 3.46% 1.61% 0.59% 0.41%0.46% 0.23% E4 17.47% 2.04% 1.12% 0.53% 0.39% 0.31% 0.27% F1 19.31%3.62% 1.51% 0.98% 0.45% 0.45% 0.55% F2 19.65% 1.94% 1.15% 0.55% 0.42%0.47% 0.40% F3 13.22% 2.38% 1.06% 0.74% 0.47% 0.36% 0.72% F4 25.18%2.07% 1.06% 0.83% 0.41% 0.29% 0.26% % Recovered Dose-FemaleAnimals-Urine per Day-Day 11 Necropsy G1  5.87% 1.01% 0.34% 0.23% 0.20%0.27% 1.00% 0.57% 0.49% 0.42% 0.53% G2 14.37% 0.65% 0.52% 0.26% 0.31%0.14% 0.87% 0.38% 0.16% 0.08% 0.30% G3 22.65% 0.74% 0.40% 0.30% 0.15%0.12% 0.52% 0.11% 0.30% 0.17% 0.25% G4 16.66% 1.36% 0.29% 0.26% 0.11%0.00% 0.48% 0.14% 0.04% 0.11% 0.17% G5 18.80% 1.18% 0.30% 0.17% 0.00%0.00% 0.32% 0.24% 0.29% 0.00% 0.38% G6 24.48% 0.80% 0.43% 0.26% 0.15%0.11% 0.50% 0.14% 0.27% 0.20% 0.18% G7 25.01% 0.51% 0.32% 0.30% 0.07%0.00% 0.33% 0.33% 0.33% 0.37% 0.24% G8 19.95% 1.02% 0.71% 0.14% 0.23%0.14% 0.75% 0.34% 0.36% 0.31% 0.33% H1 20.57% 7.92% 3.90% 3.05% 1.94%1.36% 1.74% 0.96% 0.92% 0.35% 0.20% H2 20.95% 8.51% 4.79% 3.34% 1.91%1.21% 1.68% 0.76% 0.50% 0.32% 0.84% H3 19.90% 7.65% 4.05% 2.72% 2.47%1.73% 1.50% 0.90% 1.01% 0.25% H4  1.76% 2.69% 2.57% 0.84% 1.16% 0.80%1.67% 0.53% 0.62% 0.27% 0.28% H5 20.72% 6.20% 3.66% 2.10% 1.62% 1.18%1.63% 0.64% 0.49% 0.21% 0.33% H6  6.70% 7.54% 4.86% 3.61% 3.01% 1.69%1.84% 0.85% 0.72% 0.49% 0.44% H7 19.37% 5.46% 4.80% 2.55% 2.10% 1.57%1.87% 1.18% 0.87% 0.38% 0.55% H8  2.05% 8.58% 5.85% 2.58% 2.23% 1.37%1.77% 0.65% 0.69% 0.35% 0.31% I1 19.74% 3.96% 1.65% 0.77% 0.94% 0.65%0.87% 0.32% 0.20% 0.58% 0.39% I2  6.58% 5.51% 1.23% 0.85% 0.87% 0.41%1.16% 0.70% 0.51% 0.74% 0.44% I3 15.30% 4.41% 3.48% 0.98% 0.59% 0.44%0.64% 0.71% 0.73% 0.92% 0.55% I4 28.39% 4.63% 2.00% 1.08% 0.63% 0.32%0.59% 0.52% 0.48% 0.42% 0.31% I5 24.31% 4.05% 1.41% 0.65% 0.79% 0.35%0.75% 0.77% 0.39% 0.40% 0.43% I6 21.90% 5.30% 1.65% 0.92% 0.76% 0.48%0.99% 0.79% 0.60% 0.38% 0.43% I7 24.69% 3.86% 1.46% 0.89% 0.28% 0.51%1.27% 0.51% 0.51% 0.38% 0.00% I8 20.49% 5.05% 1.93% 0.93% 1.05% 0.55%0.64% 0.79% 0.63% 0.41% 0.71% K1 22.09% 0.70% 0.23% 0.15% 0.26% 0.28%0.36% 0.26% 0.10% 0.37% 0.61% K2 18.42% 1.10% 0.29% 0.13% 0.25% 0.00%0.51% 0.41% 0.11% 0.00% 0.53% K3 21.15% 0.62% 0.11% 0.14% 0.07% 0.15%0.11% 0.30% 0.46% 0.06% 0.43% K4 23.56% 1.10% 0.49% 0.00% 0.29% 0.09%0.45% 0.29% 0.00% 0.40% 0.15% K5 21.23% 1.44% 0.39% 0.66% 0.27% 0.11%0.39% 0.29% 0.26% 0.51% 0.45% K6 11.82% 1.14% 0.37% 0.00% 0.17% 0.08%0.54% 0.39% 0.64% 0.59% 0.15% K7 23.04% 1.25% 0.78% 0.00% 0.23% 0.07%0.20% 0.45% 0.60% 0.38% 0.49% K8 22.37% 0.73% 0.58% 0.17% 0.24% 0.11%0.21% 0.15% 0.32% 0.00% 0.00% L1 12.59% 2.90% 1.04% 0.50% 0.64% 0.60%0.61% 0.30% 0.21% 0.24% 0.20% L2 16.03% 1.60% 0.84% 0.38% 0.28% 0.06%0.22% 0.16% 0.30% 0.45% 0.41% L3 21.48% 1.70% 1.21% 0.59% 0.60% 0.37%0.72% 0.05% 0.20% 0.12% 0.20% L4 17.40% 2.41% 0.62% 0.77% 0.52% 0.49%1.23% 0.55% 0.22% 0.20% 0.40% L5 21.11% 1.15% 1.08% 0.45% 0.67% 0.33%0.46% 0.30% 0.55% 0.21% 0.14% L6 22.45% 2.95% 1.06% 0.81% 0.60% 0.48%0.44% 0.60% 0.27% 0.30% 0.18% L7 21.35% 2.14% 0.54% 0.67% 0.50% 0.37%0.51% 0.16% 0.25% 0.34% 0.34% L8 20.51% 3.00% 1.49% 1.28% 0.69% 0.43%0.75% 0.62% 0.67% 0.63% 0.31% M1 19.50% 2.27% 0.81% 0.47% 0.66% 0.38%0.73% 0.40% 0.40% 0.12% 0.35% M2 24.06% 2.99% 1.49% 0.64% 0.36% 0.35%0.69% 0.36% 0.45% 0.23% 0.30% M3 17.35% 2.82% 1.40% 0.77% 0.19% 0.46%0.76% 0.42% 0.37% 0.16% 0.22% M4 21.87% 2.82% 1.64% 0.68% 0.53% 0.26%0.65% 0.32% 0.42% 0.28% 0.20% M5 18.70% 2.73% 1.14% 0.62% 0.60% 0.36%0.63% 0.43% 0.40% 0.21% 0.36% M6  2.45% 2.07% 1.43% 0.31% 0.47% 0.41%0.83% 0.34% 0.61% 0.62% 0.43% M7 28.31% 4.97% 1.07% 0.58% 0.30% 0.12%0.47% 0.74% 0.41% 0.46% 0.26% M8 25.46% 3.05% 1.00% 0.72% 0.22% 0.61%0.38% 0.33% 0.48% 0.27% 0.42% *Comments: Day 10-H3: collected by notcalculated Animal ID Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day9 Day 10 Day 11 % Recovered Dose-Male Animals-Feces per Day-Day 7Necropsy A1  4.54%  1.10%  2.13% 2.47% 1.92%  1.46% 2.17% A2 11.70% 1.31%  2.26% 1.68% 0.92%  0.80% 1.37% A3  7.30%  1.16%  1.31% 1.60%1.17%  1.11% 1.35% A4 11.62%  1.61%  0.91% 1.84% 0.72%  0.97% 0.82% B1 4.37%  7.66%  2.15% 3.14% 1.34%  2.28% 1.46% B2  8.32% 12.16%  3.85%4.08% 2.97%  6.44% 2.68% B3  7.75%  9.09%  3.59% 1.98% 0.93%  1.47%1.21% B4  4.30% 13.43%  3.55% 4.25% 2.49%  2.02% 2.10% C1  8.78% 15.31% 2.49% 3.01% 1.70%  1.91% 1.21% C2  6.29% 12.37%  5.42% 3.04% 2.00% 2.24% 1.32% C3  9.94% 22.78%  4.54% 5.87% 3.00%  2.37% 1.95% C4  4.97%14.58%  6.24% 3.77% 2.15%  2.32% 1.31% D1  9.37%  1.92%  2.05% 2.20%1.55%  1.38% 1.74% D2  6.97%  0.95%  1.02% 2.37% 1.09%  1.31% 1.13% D3 8.18%  1.35%  1.67% 1.63% 1.38%  1.64% 1.70% D4  5.47%  1.34%  1.91%3.05% 1.46%  3.11% 2.12% E1  6.19% 19.35%  3.91% 5.36% 1.97%  2.01%2.11% E2 11.96% 15.22%  2.72% 3.66% 1.84%  2.66% 1.97% E3  7.28% 16.75% 3.36% 5.40% 1.67%  2.92% 2.22% E4 11.97% 13.98%  3.87% 5.25% 1.79% 1.87% 1.18% F1  6.91% 14.25% 13.31% 4.17% 1.77%  1.60% 0.74% F2  7.78%13.00%  3.36% 4.09% 2.34%  2.84% 2.65% F3 14.62% 16.24%  3.68% 3.98%1.75%  1.72% 2.45% F4  9.13% 15.40%  5.91% 3.52% 1.78%  1.54% 1.59% %Recovered Dose-Male Animals-Feces per Day-Day 11 Necropsy G1 12.82% 2.03%  1.71% 3.33% 1.15%  3.76% 0.86% 1.89% 1.61% 1.12% 1.40% G2  7.64% 1.97%  1.46% 2.66% 1.43%  1.59% 1.69% 1.73% 1.89% 1.44% 1.24% G3  6.70% 0.82%  1.00% 1.64% 0.97%  1.31% 1.61% 1.36% 1.68% 0.88% 0.94% G4 13.99% 2.13%  2.06% 4.92% 1.86%  2.34% 1.81% 1.99% 1.85% 1.08% 1.18% G5 12.37% 1.41%  2.08% 3.61% 1.75%  2.69% 1.06% 2.43% 1.73% 1.00% 1.19% G6  8.41% 0.77%  1.13% 1.81% 0.75%  2.71% 1.07% 2.14% 0.85% 0.57% 0.63% G7  5.16% 0.84%  0.85% 1.94% 0.92%  1.59% 0.97% 1.52% 1.00% 0.95% 1.00% G8  7.26% 1.28%  2.15% 2.81% 2.34%  2.22% 1.75% 1.77% 1.13% 1.09% 1.03% H1  9.15% 2.78%  3.63% 3.78% 1.51%  1.64% 1.05% 0.81% 0.43% 0.38% 0.37% H2  8.02% 4.90%  6.69% 4.47% 1.78%  2.00% 1.04% 0.75% 0.51% 0.40% 0.35% H3  6.78% 1.40%  4.80% 3.18% 1.71%  2.45% 1.03% 1.29% 0.65% 0.33% 0.40% H4 17.54% 7.06% 10.31% 7.97% 3.62%  2.79% 2.05% 1.70% 1.29% 0.79% 0.78% H5 10.87% 3.40%  4.70% 3.38% 1.81%  1.57% 1.28% 0.85% 0.58% 0.42% 0.36% H6 15.09% 6.60%  6.91% 4.66% 1.45%  1.96% 0.78% 0.78% 0.39% 0.30% 0.30% H7  6.58% 7.14%  8.25% 5.75% 2.38%  1.83% 1.36% 1.19% 0.64% 0.52% 0.52% H8 17.21% 5.64%  7.45% 4.65% 1.92%  1.74% 3.81% 1.40% 0.87% 0.68% 0.77% I1  9.44%24.63%  7.33% 4.73% 2.15%  1.96% 1.48% 0.86% 0.53% 0.46% 0.47% I2 11.73%29.18%  7.00% 4.29% 1.67%  1.64% 2.52% 0.84% 0.71% 0.52% 0.38% I3 10.15%24.62%  5.92% 4.45% 2.13%  1.89% 0.83% 0.55% 0.43% 0.43% 0.37% I4  5.92%13.96%  6.28% 3.39% 2.90%  2.15% 1.51% 0.84% 0.64% 0.45% 0.43% I5  9.00%18.50%  9.54% 5.21% 1.73%  2.00% 0.94% 0.76% 0.57% 0.45% 0.48% I6  6.11%19.63%  7.12% 4.16% 2.06%  1.56% 1.29% 0.55% 0.47% 0.40% 0.44% I7  8.55%17.47%  7.60% 3.65% 1.56%  1.92% 1.15% 1.22% 0.49% 0.47% 0.50% I8  9.19%19.39%  4.73% 3.13% 2.26%  1.66% 1.02% 0.87% 0.53% 0.36% 0.43% K1  6.75% 1.18%  1.75% 3.06% 1.26%  1.67% 1.79% 1.63% 1.26% 1.28% 1.34% K2  5.72% 1.90%  1.65% 2.06% 1.59%  2.00% 1.49% 1.41% 0.96% 1.06% 0.97% K3  9.45% 1.18%  1.71% 2.62% 1.41%  2.07% 0.94% 1.40% 0.85% 0.77% 1.10% K4  5.19% 2.88%  1.47% 1.22% 0.85%  1.78% 1.16% 1.26% 0.88% 0.73% 1.64% K5  8.35% 1.44%  1.09% 2.86% 0.63%  1.08% 1.24% 1.29% 0.86% 0.72% 0.89% K6 14.38% 1.25%  1.84% 2.63% 1.32%  1.46% 1.46% 1.56% 1.26% 1.01% 1.18% K7  6.30% 1.06%  0.97% 2.26% 1.04%  2.46% 1.16% 1.81% 1.31% 1.12% 1.62% K8  6.81% 1.35%  1.58% 1.91% 1.65%  1.46% 1.40% 1.78% 1.57% 1.09% 1.07% L1 19.13% 9.49%  2.02% 1.98% 1.04%  3.32% 1.56% 1.03% 0.88% 0.60% 0.63% L2  9.90%10.37%  3.23% 2.69% 1.47%  1.94% 1.72% 1.27% 0.94% 0.98% 0.89% L3 13.78%11.59%  3.48% 3.71% 2.57%  2.97% 1.53% 1.54% 0.96% 0.96% 0.79% L4  9.96%15.06%  4.47% 6.81% 2.42%  2.78% 1.45% 1.58% 0.88% 0.65% 0.66% L5  8.53%13.00%  2.87% 3.67% 1.75%  2.36% 2.10% 1.33% 0.57% 0.99% 0.87% L6  6.03%13.15%  3.37% 4.04% 1.82%  3.15% 1.44% 1.26% 1.17% 1.01% 0.87% L7 11.30%16.38%  4.43% 3.55% 2.26%  2.15% 1.80% 1.23% 1.08% 1.02% 0.84% L8  9.22% 8.84%  2.71% 3.76% 1.72%  2.74% 1.29% 0.92% 0.78% 0.90% 0.66% M1  8.69%16.84%  4.32% 3.42% 1.00% 10.71% 5.02% 1.81% 1.01% 0.48% 0.57% M2  9.69%15.20%  6.13% 4.94% 2.50%  2.32% 2.31% 1.55% 1.12% 0.47% 0.46% M3 13.97%15.54%  4.17% 4.01% 1.52%  2.47% 1.09% 1.22% 0.73% 0.92% 0.82% M4  8.94%13.29%  2.65% 4.02% 1.62%  1.70% 1.15% 0.94% 0.57% 0.59% 0.41% M5  5.04%15.04%  4.16% 6.06% 1.76%  3.63% 1.51% 1.24% 1.01% 0.65% 0.70% M6 18.41%19.29%  6.33% 4.40% 1.90%  2.41% 1.35% 2.07% 0.96% 1.22% 1.12% M7  8.12%14.53%  4.24% 3.22% 1.20%  1.58% 0.86% 0.87% 0.64% 0.76% 0.68% M8  5.36%19.54%  3.82% 2.93% 1.08%  1.99% 1.01% 1.10% 0.76% 0.85% 1.01%

In EXAMPLE 5-EXAMPLE 9 the treatment dose levels for each in vivo studyare presented in μmol/kg and/or mg/kg. Based on the molecular weight of750.71 g/mol for the API 3,4,3-L1(1,2-HOPO), the mg/kg dose level isdivided by 0.7507 to obtain the dose level in μmol/kg, while the μmol/kgdose level is multiplied by 0.7507 to obtain the dose level in mg/kg.Dose levels commonly used in the nonclinical studies are displayed asboth μmol/kg and mg/kg in TABLE 0.1 for reference purposes.

TABLE 0.1 COMMONLY USED NON-CLINICAL DOSE LEVELS OF 3,4,3-LI(1,2-HOPO)IN μMOL/KG AND MG/KG Dose Level (μmol/kg) Dose Level (mg/kg) 1 0.8 3 2.310 7.7 30 22.5 50 37.5 60 45.1 100 75.1 200 150 300 225 400 300 532 400600 451 1000 751 1800 1352

Example 5—Radiolabeled In Vivo ADME Studies in Rodents

Formulated material was used in the single dose in vivo ADMEcharacterization studies in Sprague Dawley rats and Swiss-Webster micewith 14C-labeled 3,4,3-L1(1,2-HOPO).

Pharmacokinetic parameters and disposition/biodistribution of3,4,3-L1(1,2-HOPO) were characterized in vivo (with the aid of two 14Clabels on the spermine backbone of the parent product) in two non-GLPstudies using Swiss-Webster mice and Sprague Dawley rats. The studydesigns for these two studies are shown in TABLE 5.1 and TABLE 5.2,respectively. Groups of six mice (3 male, 3 female) were given a singledose of [14C]-3,4,3-L1(1,2-HOPO) via iv, ip, or po routes, respectively.An additional group of six mice (3 male, 3 female) were administered the14C-labeled API with permeability enhancer (10% sodium oleate) by oralgavage (po). Similarly, groups of six rats (3 male, 3 female) wereadministered a single iv dose of the 14C-labeled API or a single po doseof the 14C-labeled API with 10% sodium oleate. In each of these studies,samples were collected at scheduled time points up to 24 hr post-doseand analyzed for 14C content using liquid scintillation counting. Blood,liver, kidney, feces and urine were collected and analyzed from themice. Blood, brain, liver, kidney, lungs, spleen, skeletal muscletissues, gastrointestinal (GI) tract samples, carcasses, feces, andurine were collected and analyzed from the rats.

TABLE 5.1 STUDY DESIGN FOR RADIOLABELED IN VIVO ADME STUDIES IN MICEDose Dose Necropsy Level Level ¹⁴C Dose No. of Time Group Route(μmol/kg) (mg/kg) (μCi/kg) Formulation Mice (min or hr) 1 iv 50 37.5 50— 18M 5, 15, 30 min; 2 iv 100 75.1 100 — 18F 1, 4, 24 hr 3 ip 50 37.5 50— 18M 4 ip 100 75.1 100 — 18F 5 oral 100 75.1 100 — 18M/18F 30, 45 min;1, 2, 6, 24 hr 6 oral 100 75.0 100 10% sodium 18M/18F 10, 20, 30, 45,oleate 60, 120 min

TABLE 5.2 STUDY DESIGN FOR RADIOLABELED IN VIVO ADME STUDY IN RATS DoseDose Necropsy Level Level ¹⁴C Dose No. of Time Group Route (μmol/kg)(mg/kg) (μCi/kg) Formulation Rats (hr) 1 iv 100 75.1 200 — 3M/3F 2 23M/3F 6 3 3M/3F 24 4 oral 100 75.1 200 10% sodium 3M/3F 2 5 oleate 3M/3F6 6 3M/3F 24

The pharmacokinetic parameters determined from these studies arepresented in TABLE 5.3. Blood was collected at 6-8 time points from 5min to 24 post-dose. Plasma concentration-time profiles showed similarlog-linear decays after iv administration in mice and rats, and theradiolabeled compound was rapidly distributed throughout theextracellular fluid space with higher peak concentrations and totalplasma exposure in rats (C0=463 and 422 Pg-eq/ml, AUC=354 and 211 hrPg-eq/ml for male and female, respectively) than in mice (C0=342 and 76Pg-eq/mL, AUC=66.2 and 41.7 hr Pg-eq/ml for female and male,respectively). Radioactivity was cleared from plasma with t½ values ofapproximately 1.6 and 8 hr for mice and rats, respectively, after ivadministration. Intraperitoneal administration of[14C]-3,4,3-L1(1,2-HOPO) in mice resulted in a lower level ofradioactivity in plasma than through the iv route, but significantlyhigher than by the po route. The oral bioavailability (F) of3,4,3-L1(1,2-HOPO) was limited, as indicated by its low plasma exposure.Bioavailability of the radioactive compound was slightly higher infemales compared with males (1.2% vs. 2.6% in male and female mice,respectively; 0.4% vs. 1.1% in male and female rats, respectively), asshown in TABLE 5.3. Formulation of 3,4,3-L1(1,2-HOPO) with sodium oleateresulted in a moderate improvement in systemic exposure: The Cmaximproved by approximately 3-fold from 0.32 to 0.93 Pg-eq/ml in male and0.55 to 1.4 Pg-eq/ml in female mice. In addition, the AUC, whencalculated over 2 hr posttreatment, increased from 8.3±6.2 to 17.4±6.7min Pg-eq/ml in males and 23.0±15.4 to 35.1±18.9 min Pg-eq/ml infemales, which translates into an oral bioavailability improvement ofabout 2-fold in mice.

TABLE 5.3 MEAN PHARMACOKINETIC PARAMETERS OF RADIOACTIVITY FOR MALE ANDFEMALE SWISS-WEBSTER MICE AND SPRAGUE DAWLEY RATS ADMINISTERED[₁₄C]-3,4,3-LI(1,2-HOPO) Sex Dose C₀ ^(a) or C_(max) T_(max) AUC^(b)t_(1/2) F^(c) Route Species (n = 2-3) (μmol/kg) (μg-eq/ml) (h) (hμg-eq/ml) (h) (%) iv Mouse Male 50 76 ± 47  NA^(d) 41.7 ± 3.2  1.6 ± 0.5NA Female 100 342 ± 211 NA 161.6 ± 21.5  1.6 ± 0.3 NA Rat Male 100 463NA  354 ± 26.9 8.1 NA Female 100 422 NA  211 ± 28.7 8.6 NA ip Mouse Male50 16.4 ± 7.1  0.33 ± 0.14 39.8 ± 10.5 NA NA Female 100 61.7 ± 36.7 0.33± 0.14 187.5 ± 14   NA NA po Mouse Male 100 0.32 ± 0.23 0.63 ± 0.18 1.94± 2.80  NC^(e) 1.2 Female 100 0.55 ± 0.29 0.67 ± 0.29 3.93 ± 3.17 NC 2.6Rat Male 100 0.29 ± 0.20 0.67 1.30 ± 0.37 NC 0.4 Female 100 0.15 ± 0.040.68 2.40 ± 0.44 NC 1.1 ^(a)C₀ is the plasma concentration extrapolatedto time zero. ^(b)AUC presented is calculated to the last data point at24 h. ^(c)The bioavailability F is calculated using the formula:[(Dose_(iv) × AUC_(po))/(Dose_(p0) × AUC_(iv))] × 100% ^(d)NA = notapplicable. ^(e)NC = not calculated; insufficient data for parameterestimation.

Analysis of radioactivity levels in tissues showed that radioactivityfrom [₁₄C]-3,4,3-L1(1,2-HOPO) was distributed rapidly into the liver andkidneys following an iv injection. In general, the highest level ofradioactivity in kidneys and liver was detected early at 1 hr in miceand 2 hr in rats post-dose (FIG. 19A-FIG. 19F and FIG. 20A-FIG. 20D). Asimilar trend was observed in mice injected ip, in which the highestlevel of radioactivity in kidneys and liver was detected at 1 hrpost-dose. No major differences in the radioactive uptake into liver andkidneys were observed between iv and ip routes of administration inmice, indicating that both routes are effective for[₁₄C]-3,4,3-L1(1,2-HOPO) distribution. In the iv treatment group forrats, the general rank order of tissue concentrations at the 2 hr timepoint was kidney>liver>lung>brain spleen muscle. Liver and kidney tissueradioactivity levels were similar and remained high at all the timepoints after iv administration, whereas levels in other tissuesdecreased more rapidly after 2 hr. As a result, after the iv dose,radioactivity tended to concentrate in the elimination organs (kidneyand liver); thus tissue-to-plasma ratios increased with time in thesetissues. In contrast, after the po dose, the highest concentrations ofradioactivity were observed in the feces and GI tract, and very lowlevels of radioactivity were observed in urine, plasma, and the othertissues (FIG. 19A-FIG. 19F) and FIG. 20A-FIG. 20D).

The principal excretion routes of radioactivity from[14C]-3,4,3-L1(1,2-HOPO) were both the feces and urine after an iv dosebased on high levels of radioactivity in both feces and urine as well assignificant radioactivity in the excretory tissues, kidney, liver and GItract. Fecal elimination by 24 hr post-dose accounted for approximately62% and 16% of the iv administered dose in mice and rats, respectively.Urinary excretion accounted for approximately 12-23% of the ivadministered dose in these rodents with renal excretion of 14C beginningas early as 5 min after iv injection in mice. In contrast, followingoral administration, excretion was primarily by the fecal routeaccounting for approximately 89% and 41% of the orally administered dosein mice and rats, respectively, by 24 h post-dose. Urinary excretionaccounted for <1% of the orally administered dose and low radioactivitylevels were detected in systemic circulation and in tissues except forthe GI tract. In mice, the highest accumulation of 14C was seen in thefeces after all three administration routes (i.e., iv, ip and po)confirming that the biliary pathway is the main mode of elimination atleast for the iv and ip administration routes. Based on the colonictransit time in rats of 15.5 hr, 11 the radioactivity found in the fecesin the first 24 hr after po administration is most likely unabsorbedcompound. Although hepatic metabolism followed by excretion in the bileis possible for 3,4,3-L1(1,2-HOPO) that is orally bioavailable, the verylow level of radioactivity in the blood and tissues in the po groupanimals suggests that the predominant route of elimination of an oraldose of 3,4,3-L1(1,2-HOPO) is through the feces and is comprised ofunabsorbed parent compound and metabolite(s) resulting from the firstpass effect of the liver or biotransformations in the gut. Metaboliteprofiling of [14C]-3,4,3-L1(1,2-HOPO) was performed using an HPLC methodon selected urine, feces, kidney, liver, and lung samples from theSprague Dawley rat. Only samples containing the highest totalradioactive levels were selected for analysis. A total of 11 peaks weredetected, of which 6 were from uncharacterized interactions between thetest article and matrix components because they were also seen in spikedblank matrix controls. These 6 peaks are not metabolites and areconsidered alternate forms of the parent compound (e.g. complexes of thetest article with metallic ions). The 5 other radioactive species (peaksP1-P4 and P10) are considered potential metabolites. Feces-specificmetabolite peaks P2, P3, and P10 represented a combined 10.5-11.4% and0.5-4.2% of the administered dose after po or iv administration,respectively, in rats. Peak P10 was the most abundant peak in allsamples analyzed. It represented up to 10% of the administered dose infeces after po administration while the other two feces-specific peaksrepresented <1% of the administered dose after po administration. PeakP10 was also the predominant, and sometimes the only, peak in fecessamples after iv administration. P10 could be of biliary origin, or,more likely, it could be the product of a transformation within theintestinal tract, either through a spontaneous degradation process ormediated by the intestinal flora, since it was not detected in liversamples. P1 was the only metabolite peak identified in urine,representing ≤0.4% of the iv administered dose, and was absent in thesingle urine sample analyzed after po administration. In conclusion,evaluation of the metabolite profiles demonstrated that a putative majormetabolite of [14C]-3,4,3-L1(1,2-HOPO) is formed (P10) that accounts for˜10% of an administered oral dose. Thus it is likely that the observedlow bioavailability of [14C]-3,4,3-L1(1,2-HOPO) is due both tobiotransformation processes, likely in the gut, as well as relativelylow absorption after oral administration.

Example 6—GLP Single Dose Oral Safety with Pharmacokinetic AssessmentStudy in Beagle Dogs

Pharmacokinetic parameters in beagle dogs after a single oraladministration of formulated capsules was determined in a GLP study. Avalidated bioanalytical method was used to determine the plasmaconcentration in 3 dogs/sex after capsule administration of 37.5, 75,and 150 mg/kg (50, 100, and 200 μmol/kg) of clinically formulated3,4,3-L1(1,2-HOPO). Plasma concentrations at all three dose levelspeaked at 0.6-1.1 hr post-dose (Tmax), and trended higher in females(FIG. 21B) compared with males (FIG. 21A). Similarly, mean exposure,based on AUCinf, was 1979±777 hr·ng/ml and 4741 hr·ng/ml for low-dosemales and females, respectively; 4317±1721 hr·ng/ml and 8610 hr·ng/mlfor mid-dose males and females, respectively; and 12022±5458 hr·ng/mland 8305±1607 hr·ng/ml for high-dose males and females, respectively.Mean Cmax and mean AUCinf values increased relatively proportional todose (all doses in males, low to mid doses in females) and trendedapproximately 2-fold higher in females compared with males at the lowand mid doses (1.7- to 2.4-fold higher). The mean t½ was consistentlyshort across dose groups and ranged from 0.5 to 0.9 hr.

The pharmacokinetic parameters in two non-GLP 7-day repeat dose studiesin beagle dogs (SRI No. B677-13 with formulated capsules and SRI No.M835-11 with oral gavage delivered API) were in general agreement withthe single dose GLP study and showed that 3,4,3-L1(1,2-HOPO) did notaccumulate in the plasma after 7 days of dosing. These two pilot studiesincluded an iv administration group so that oral bioavailability couldbe calculated. The oral bioavailability was low at <3% and essentiallythe same in both studies regardless of whether the dogs receivedformulated capsules or 3,4,3-L1(1,2-HOPO) dissolved in PBS withoutsodium oleate. One male and two female dogs were administered 37.6 mg/kg(50 μmol/kg) by the iv route which resulted in an observed peak plasmalevel of 115±11 μg/ml, mean AUCinf of 64 hr·μg/ml and a t½ value of 0.4hr. Volume of distribution was 0.3 L/kg, consistent with drug that isdistributed primarily to the extracellular space. The clearance (Cl) was594 ml/hr/kg with an indication that plasma clearance may becomesaturated at higher plasma concentrations.

Example 7—Conclusions of Pharmacokinetic and ADME Studies from Examples5 and 6

The pharmacokinetics of 3,4,3-L1(1,2-HOPO) were generally similar acrossspecies. Oral bioavailability of formulated and unformulated3,4,3-L1(1,2-HOPO) was low at <3% in the dog and the bioavailability oftotal radioactivity from [₁₄C]-3,4,3-L1(1,2-HOPO) in mice and rats wasalso <3%. Formulation of API with sodium oleate enhanced exposureparameters by approximately 2- to 3-fold in mice. When administeredorally, 3,4,3-L1(1,2-HOPO) was eliminated almost entirely through thefecal route either as unabsorbed parent compound or as metabolite(s)formed either by the liver or in the small intestine. Evaluation of themetabolite profiles in rats demonstrated that a putative majormetabolite of [₁₄C]-3,4,3-11(1,2-HOPO) is feces-specific and that itaccounts for ˜10% of an administered oral dose. This metabolite could beof biliary origin, or, more likely, it is the product of metabolismwithin the intestinal tract, either through a spontaneous degradationprocess or mediated by the intestinal flora. Thus it is likely that theobserved low bioavailability of [₁₄C]-3,4,3-L1(1,2-HOPO) is due both tobiotransformation processes as well as relatively low absorption of anoral dose.

When administered to rodents by the iv route, 3,4,3-L1(1,2-HOPO) wasdistributed rapidly into the liver and kidneys and eliminated by boththe renal and biliary routes. The compound appears to be metabolized inthe GI tract, degraded slightly in gastric fluid, and stable in plasma.

Plasma concentrations after an oral dose peaked (Tmax) at similar timespost-dose in rodents and with the clinical formulation in dogs (˜0.7 hrand 0.6-1.1 hr post-dose in rodents and dogs, respectively). Mean t½after oral administration was consistently short across dose groups andspecies at ˜1 hr in rats and 0.5 to 0.9 hr in dogs. Cmax, AUC, and oralbioavailability trended ˜2-fold higher in females than males in allthree species (mice, rats, and dogs) and increased relativelyproportional to dose. When co-formulated with sodium oleate as apermeability enhancer, the labeled [14C]-3,4,3-L1(1,2-HOPO) displayed anincreased exposure that translates into an oral bioavailabilityimprovement of about 2- to 3-fold in mice. In beagle dogs administeredthe clinical formulation of 3,4,3-L1(1,2-HOPO), exposure based on Cmaxand AUCinf values increased relatively proportional to dose (all dosesin males, low to mid doses in females) and trended higher in femalescompared with males at the low and mid doses (1.7- to 2.4-fold higher).The mean t½ was consistently short across dose groups and ranged from0.5 to 0.9 hr.

Plasma protein binding differed across species; it was greatest in dog(95%), intermediate in human (29%), and lowest in rat (5%) when testedat 10 μg/ml. In vitro human liver microsome experiments showed that thecompound was relatively stable, and these results are consistent withthe relatively low extent of metabolism observed in vivo in the rat.3,4,3-L1(1,2-HOPO) did not inhibit the activity of CYP1A2, CYP2B6,CYP2C₉, CYP2C19, CYP2D6, and CYP3A4 and thus is unlikely to be a sourceof drug interactions for other agents metabolized by these enzymes.

Example 8—Single Oral Dose GLP Safety Study in Beagle Dogs

The clinical formulation was tested in a single dose GLP toxicology andcardiovascular safety pharmacology study in beagle dogs. The studydesign is presented in TABLE 8.1. This study demonstrated a NOAEL of37.5 mg/kg (50 μmol/kg) 3,4,3-L1(1,2-HOPO) in dogs following oralcapsule administration of formulated material at 37.5, 75, or 150 mg/kg(50, 100, and 200 μmol/kg).

TABLE 8.1 STUDY DESIGN FOR GLP SINGLE DOSE ORAL SAFETY STUDY IN BEAGLEDOGS Dose Dose No. of Dogs No. of Dogs Level Level No. of SacrificedSacrificed Group Treatment (μmol/kg) (mg/kg) Dogs on Day 2 on Day 15 1Empty Capsule 0 0 6M/6F 3M/3F 3M/3F 2 Formulated 3,4,3- 50 37.5 6M/6F3M/3F 3M/3F LI(1,2-HOPO) in Capsule 3 Formulated 3,4,3- 100 75 6M/6F3M/3F 3M/3F LI(1,2-HOPO) in Capsule 4 Formulated 3,4,3- 200 150 6M/6F3M/3F 3M/3F LI(1,2-HOPO) in Capsule

Based on this study, the MTD in dogs after a single oral administrationis considered to be greater than 150 mg/kg. A11 dogs (6/sex/group; total48) survived to their scheduled sacrifice on Day 2 or 15, andadministration of 3,4,3-L1(1,2-HOPO) produced no meaningful changes inbody weight, food consumption, ophthalmology, cardiovascularassessments, clinical pathology, urinalysis, gross necropsyobservations, or organ weights.

Clinical observations that were associated with test articleadministration included post-dose diarrhea and emesis. Specifically,dogs in the mid- and high-dose groups experienced slight or extremediarrhea on Day 1 between 1 and 6 hr post-dose. Diarrhea was absent fromthe lowdose group. Slight diarrhea was seen in 17% of males and 33% offemales in the high-dose group and in 67% of females in the mid-dosegroup. Extreme diarrhea was seen in 33% of highdose males. By Day 2, 8of the 9 affected dogs were normal, with the exception of one highdosegroup female that had continued slight diarrhea. Slight to moderateemesis occurred <2 hr post-dose (and oftentimes <1 hr post-dose) in 1-3dogs from each of the 3,4,3-L1(1,2-HOPO) treatment groups and in none ofthe vehicle control-treated dogs. Post-dose emesis appeared to bedose-dependent since it occurred in 0, 1, 2, and 3 dogs out of 12 dogsper group in control, low, mid, and high dose groups, respectively.Post-dose emesis is a common response to oral dose administration indogs, and a single occurrence of it in the low dose group could betreatment-related rather than test article-related and is not considereda dose-limiting event. Slight post-dose emesis in this one male dog wasthe only finding present in the 37.5 mg/kg lowdose treatment group.Similar dose dependent, transient emesis and diarrhea were observed ˜1hr post-dose in the non-GLP repeat dose beagle dog study (SRI No.B677-13) where 2 dogs/sex were administered formulated capsules oncedaily for 7 days at doses of 75.1, 150, or 300 mg/kg (100, 200, and 400μmol/kg). However, soft stools or diarrhea were not observed in SpragueDawley rats after oral gavage administration of 400-1300 mg/kg (532-1732μmol/kg) for 7 days (SRI No. M801-10) or 7.7-76.9 mg/kg (10-102 μmol/kg)for 28 days (SRI No. M512-07).

Renal findings of diffuse, moderate, proximal tubular casts anddilation; moderate interstitial hemorrhage; and mild interstitialhemosiderin pigmentation were observed at the Day 15 recovery sacrificein 1 of 3 female dogs in the high-dose group (150 mg/kg). No similarrenal histopathology findings were found in any of the other on-studydogs. The observation of hemosiderin without fibrosis in the kidneysections from this one dog is consistent with the renal findings havingdeveloped within one to three days prior to the Day 15 necropsy. Thisdog also had corresponding small increases (1.8- and 1.3-fold,respectively) in the renal function markers BUN and CR on Day 15relative to pre-test. The timing of the renal findings in this recoverygroup dog is surprising given the fact that 3,4,3-L1(1,2-HOPO) wasadministered only on Day 1. Therefore, it is unclear if these renalfindings are related specifically to high-dose administration of3,4,3-L1(1,2-HOPO). There were no toxicologically meaningfulhistopathology findings in any of the dogs on Day 2 or in any of theother dogs on Day 15. Similar renal findings were also absent from thenon-GLP 7-day repeat dose dog study (SRI No. B677-13) where 2 dogs/sexreceived formulated material at higher doses for 4 days followed byequivalent doses for 3 days and then were sacrificed on Day 8. Therewere also no renal findings in the Sprague Dawley rat studies after 7and 28 days of oral gavage dose administration (SRI Nos. M801-10 andM512-07, respectively).

An evaluation of the serum levels of iron, unsaturated iron bindingcapacity, total iron binding capacity, magnesium, and ferritin wereincluded in the clinical pathology analysis during the dog safetystudies because 3,4,3-L1(1,2-HOPO) is a potent metal chelator. In theGLP study with 3 dogs/sex, none of these parameters were meaningfullyaltered with statistical significance on Days 2 or 15 in treated groupswhen compared with controls although the standard deviations were largeand the number of dogs per group was small. In the non-GLP pilot repeatdose study with even smaller numbers (2 dogs/sex) and no control group,there were indications that serum total iron levels increased ˜2-foldafter treatment when compared with pretest levels while the unsaturatediron binding capacity decreased 16-62% suggesting that the increasediron was bound to transferrin, not 3,4,3-L1(1,2-HOPO). Thus, the resultsfrom the nonclinical dog studies suggest that serum iron and magnesiumlevels are not meaningfully altered after 3,4,3-L1(1,2-HOPO)administration although these parameters are being evaluated in theclinical trial.

Example 9—GLP Single Oral Dose GLP Safety with Cardiovascular AssessmentStudy in Beagle Dogs

Cardiovascular parameters in beagle dogs after a single oraladministration of formulated capsules was determined in a GLP study

Electrocardiograms and blood pressure were evaluated from 3 male and 3female beagle dogs per dose group (0, 37.5, 75, or 150 mg/kg) atpretest, 1 and 4 hours post-dose, and 7 days after a single oral dose offormulated material (TABLE 8.1). There were no electrocardiographic,heart rate, or blood pressure findings that were attributed toadministration of 3,4,3-L1(1,2-HOPO). Instances of increased ordecreased blood pressure (hypertension or hypotension) in various dogswere considered sporadic or due to stress, excitement, or strugglingduring the time of recording and not test article related. In summary,electrocardiogram and blood pressure assessments in beagle dogs raisedno cardiovascular safety concerns.

Example 10—Permeability Enhancement of 3,4,3-L1(1,2-HOPO) Summary

The objective of the analytical study described in Part C of this reportwas to assess the feasibility of enhancing the permeability of theactive pharmaceutical ingredient 3,4,3-L1(1,2-HOPO) using oralpermeation enhancers. Evaluation was performed using an in vitro pKassay based on the Double-Sink™ PAMPA technology developed by pION, Inc.

Fifteen different permeation enhancers were evaluated for their abilityat increasing the permeability of 3,4,3-L1(1,2-HOPO), using an in vitroPAMPA assay with artificial GIT lipid membranes. A significant increasewas observed in permeability for one formulation containing 10 mg/mL ofPolysorbate 80 and 1 mg/mL of API. A11 other tested formulations showedno or minor improvement in permeability. Formulations containingPolysorbate 80 will be evaluated further in in vivo studies.

1. Purpose of Study

The purpose of this study was to provide data that can be used tosupport research efforts. It was not conducted in accordance with U.S.Food and Drug Administration (FDA) “Good Laboratory Practice forNonclinical Laboratory Studies” (GLP) regulations, as described in 21CFR Part 58. However, the study was planned, performed, recorded, andreported in accordance with standard practices to ensure data qualityand integrity.

2. Objective of Study

The objective of this study was to assess the feasibility of enhancingthe permeability of the active pharmaceutical ingredient3,4,3-L1(1,2-HOPO) using oral permeation enhancers. Evaluation wasperformed using an in vitro pK assay based on the Double-Sink™ PAMPAtechnology developed by pION, Inc.

3. Experimental Design

The permeability enhancement study was performed in two stages. In thefirst stage (TABLE 10.1), 15 formulations were prepared and screened.The second stage (TABLE 10.2) was performed to refine the concentrationsof the permeability enhancers that seemed to display permeabilityenhancement in the first screening. The screening conditions for bothstages are listed below. The sample solutions were stored throughout thestudy in 20 mL clear scintillation glass vials with polypropylene capand pulp foil liner, wrapped in aluminum foil.

TABLE 10.1 PAMPA SCREENING STAGE I PAMPA Screening Stage 1 PermeationEnhancer Formulation Vehicle Preparation Max. IIG Enhancer's API's limitor Target Target Vehicle Formulation Enhancer Literature Conc ConcVolume Code Class Selected report (mg/mL) (mg/mL) (mL) F0 (Control)3,4,3-LI(1,2- None NA NA 1 10 HOPO) API F1 Anionic Sodium lauryl  0.010%0.10 1 10 Surfactant sulfate (SLS) F2 Nitrogen Caprolactam 1.00% 2.50 110 containing Ring F3 Nonionic Polysorbate 80 20 mg/mL 2.50 1 10Surfactant F4 Bile Salts Sodium 30 mg/mL 2.50 1 10 deoxycholoate F5Fatty Esters Isopropyl myristate 1.00% 2.50 1 10 F6 Nitrogen1-Phenylpiperazine 1.00% 2.50 1 10 containing Rings F7 Natural Piperine1.00% 2.50 1 10 F8 Others Menthone 1.00% 2.50 1 10 F9 Lipid LabrafacLipophile 159 mg 5.00 1 10 WL 1349 F10 Lipid Gelucire 44/14 218 mg 5.001 10 F11 Lipid Labrafil M2130 CS 218 mg 5.00 1 10 F12 Lipid LabrafilM2125 CS 300 mg 5.00 1 10 F13 Lipid Maisine 35-1 344 mg 5.00 1 10 F14Lipid Peceol 33.3   5.00 1 10 F15 Lipid Labrasol 70 mg 5.00 1 10

TABLE 10.2 PAMPA SCREENING STAGE 2 PAMPA Screening Stage 2 PermeationEnhancer Formulation Vehicle Preparation Max. IIG Enhancer's API's limitor Target Target Vehicle Formulation Enhancer Literature Conc ConcVolume Code Class Selected report (mg/mL) (mg/mL) (mL) F0 Repeat3,4,3-LI(1,2- NA 1 10 HOPO) API F3 Repeat Nonionic Polysorbate 80 20mg/mL 2.50 1 10 F3A Surfactant 5.00 1 10 F3B 10.00 1 10 F4 Repeat BileSalts Sodium 30 mg/mL 2.50 1 10 F4A deoxycholate 10.00 1 10 F5 RepeatFatty Esters Isopropyl 1.00% 2.50 1 10 F5A myristate 10.00 1 10 F10Repeat Lipid Gelucire 44/14 218 mg 5.00 1 10 F10A 20.00 1 10 F13 RepeatLipid Maisine 35-1 344 mg 5.00 1 10 F13A 10.00 1 10 F13B 20.00 1 10 F14Repeat Lipid Peceol 33.3   5.00 1 10 F14A 10.00 1 10 F14B 20.00 1 104. Materials And Methods

a. Test and Control Articles

-   Test Article: 3,4,3-L1(1,2-HOPO)-   Manufacturer: Ash Stevens, Inc. (Detroit, Mich.)-   Lot Number: ML-11-276-   Physical Description: Pale yellow solid-   Storage Conditions: Refrigerated 2-8° C. protected from light.    Materials:

Purified Water HPLC Grade-Supplier: Ricca Sodium lauryl sulfate (SLS)Spectrum chemicals Caprolactam Spexcertiprep Polysorbate 80 (Tween 80)Spectrum chemicals Sodium deoxycholate Sigma-aldrich Isopropyl myristateSigma-aldrich 1-Phenylpiperazine Sigma-aldrich Piperine Sigma-aldrichMenthone TCI Labrafac Lipophile WL 1349 Gattefosse Gelucire 44/14Gattefosse Labrafil M2130 CS Gattefosse Labrafil M2125 CS GattefosseMaisine 35-1 Gattefosse Peceol Gattefosse Labrasol Gattefosse GIT-0lipid pIOn, Inc. Acceptor Sink Buffer pIOn, Inc. Prisma ™ Buffer pIOn,Inc. DMSO HPLC Grade, Burdick and Jackson Stirring device Gut-Box ™,pION, Inc.

-   Test Solutions: Vehicles containing the permeation enhancers were    prepared by weighing the adequate quantity of enhancer and    dissolving it into 100 mL of purified water to reach the designated    concentration. Test solutions were then prepared by weighing 10 mg    of 3,4,3-L1(1,2-HOPO), dissolving it into 10 mL of each of the    vehicles to reach a concentration of 1 mg/mL (purified water was    used for the control solution). The final pH and clarity of each    solution were recorded.

b. Sample Characterization

Visual Observation: For each sample solution, visual observationconsisted in recording color and clarity.

pH Record: The pH of each sample solution prepared for permeabilityanalysis was measured and recorded.

c. Permeability Assay

In vitro PK assay based on Double-Sink™ PAMPA assay layout:

The PAMPA Evolution96™ instrument was used for the liquid handling, UVdata collection and results processing. The system consisted of a96-well Double-Sink PAMPA Sandwich with pre-loaded stirrers. A PAMPAsandwich was formed such that each composite well was divided into twochambers, separated by a 125 μm microfilter disc (0.45 μm pores), coatedwith Pion GIT-0 phospholipids mixture. Formulations were suspended inPrisma™ buffer. GIT-0 lipid painted on a filter support created anartificial membrane separating two chamber of the permeation systemwhile the free of drug Acceptor Sink Buffer (ASB, pH 7.4) was placed inthe receiving compartment.

After introducing the formulations in the donor compartments, the PAMPAsandwich was incubated for 15-30 min or up to 24 hours and only the UVspectra of the receiver were collected. Calibrated for in vivoconditions, individual-well stirring was provided by the Gut-Box™ (PionInc.).

The appearance rate of a compound in the receiving compartment of thePAMPA Sandwich containing formulation in the donor compartment wascompared to the corresponding rate in a formulation-free system. Theratio between these two rates was reported as a Flux Ratio.

Sample Mapping Schemes (Table 10.3 and Table 10.4)

TABLE 10.3 Stage 1 Screening: 1 2 3 4 5 6 7 8 9 10 11 12 A Formulation 1Vehicle 1 Formulation 9 Vehicle 9 B Formulation 2 Vehicle 2 Formulation10 Vehicle 10 C Formulation 3 Vehicle 3 Formulation 11 Vehicle 11 DFormulation 4 Vehicle 4 Formulation 12 Vehicle 12 E Formulation 5Vehicle 5 Formulation 13 Vehicle 13 F Formulation 6 Vehicle 6Formulation 14 Vehicle 14 G Formulation 7 Vehicle 7 Formulation 115Vehicle 15 H Formulation 8 Vehicle 8 Formulation 0-Control Vehicle0-Control

TABLE 10.4 Stage 2 Screening: 1 2 3 4 5 6 7 8 9 10 11 12 A Formulation 3F3 Vehicle Formulation 14B F14B Vehicle B Formulation 3A F3A VehicleFormulation 4 F4 Vehicle C Formulation 3B F3B Vehicle Formulation 4A F4AVehicle D Formulation 13 F13 Vehicle Formulation 5 F5 Vehicle EFormulation 13A F13A Vehicle Formulation 5A F5A Vehicle F Formulation13B F13B Vehicle Formulation 10 F10 Vehicle G Formulation 14 F14 VehicleFormulation 10A F10A Vehicle H Formulation 14A F14A Vehicle Formulation0-Control F0-Control Vehicle5. Results

a. PAMPA Assay Results

Observations (formulation appearance and pH) and PAMPA permeationresults are summarized in TABLE 10.5 below for both screening stages.Based on the data obtained from the permeation assay, the GITlipid-covered membranes were stable in the presence of all testedformulations and formulation vehicles, and no leakage was detected. TheAPI 3,4,3-L1(1,2-HOPO) indicated very low permeability, comparable oreven lower than the permeability level of the references compoundRanitidine.

TABLE 10.5 PAMPA SCREENING RESULTS STAGES 1 AND 2 Formulation VehicleObservations/Results Preparation & pH API's Concentration: 1 in 100 mLPurified Water mg/mL, 10 mL Vehicle Enhancer's Flux Ration Target (Invitro pK Formulation Enhancer Conc Quantity PAMPA Code Selected (mg/mL)(mg) PH Appearance pH Assay) F0 (Control) None NA NA 7.21 Clear 3.731.00 solution F0 Repeat NA NA 7.20 Clear 3.70  1.00 ± 0.09 solution F1Sodium lauryl 0.10 10 6.58 Clear 3.74 NA sulfate solution F2 Caprolactam2.50 250 6.68 Clear 3.72 NA solution F3 Polysorbate 80 2.50 250 6.07Clear 3.74  2.22 ± 0.96 solution F3 Repeat 2.50 250 6.34 Clear 3.70 0.77 ± 0.08 solution F3A 5.00 500 6.30 Clear 3.83  0.94 ± 0.09 solutionF3B 10.00 1000 6.54 Clear 3.72 75.57 ± 5.22 solution F4 Sodium 2.50 2507.63 White 6.85  1.04 ± 0.43 deoxycholate Dispersion F4 Repeat 2.50 2507.47 White 7.08 NA Dispersion F4A 10.00 1000 7.82 Opaque 7.07 NAdispersion F5 Isopropyl 2.50 250 6.55 Clear 3.77  1.06 ± 0.72 myristatesolution F5 Repeat 2.50 250 6.73 Clear 3.79  1.86 ± 0.22 solution F5A10.00 1000 6.82 Clear 3.72  1.39 ± 0.14 solution with oily drops F6 1-2.50 250 9.99 Clear 9.12 NA Phenylpiperazine solution F7 Piperine 2.50250 6.93 Clear 3.79 NA solution F8 Menthone 2.50 250 6.76 Clear 3.72 NAsolution F9 Labrafac 5.00 500 5.73 Oily globules 3.75 NA Lipophile WLF10 Gelucire 44/14 5.00 500 3.87 Clear 3.62  0.84 ± 0.45 solution F10Repeat 5.00 500 4.17 Clear 3.72 NA solution F10A 20.00 2000 3.41 Opaque3.44  0.77 ± 0.09 dispersion F11 Labrafil M2130 5.00 500 5.65 White 3.73NA CS Dispersion F12 Labrafil M2125 5.00 500 6.16 White 3.79 NA CSDispersion F13 Maisine 35-1 5.00 500 6.23 Opaque 3.74  2.30 ± 0.93dispersion F13 Repeat 5.00 500 6.37 Opaque 3.67  0.71 ± 0.12 dispersionF13A 10.00 1000 6.35 White 3.68  0.95 ± 0.07 Dispersion F13B 20.00 20006.41 Opaque 3.92  1.04 ± 0.08 dispersion F14 Perceol 5.00 500 5.22 Clear3.70  1.18 ± 0.60 solution F14 Repeat 5.00 500 6.03 Clear 3.74  1.15 ±0.12 solution F14A 10.00 1000 6.08 Clear 3.7  0.97 ± 0.30 solution F14B20.00 2000 6.03 Clear 3.83  1.63 ± 0.43 solution F15 Labrasol 5.00 5005.27 White 3.71 NA Dispersion

For Formulations 1, 2, 4A, 8, 9, 11, 12, and 15, the UV-Visible signalin the acceptor compartment was below the detection limit and the fluxratio could not be determined. Formulations 6 and 7 showed very highpenetration rates for their corresponding vehicles that completelysaturated the UV-Visible signal in the acceptor compartment, precludingthe signal detection of the API under the strong vehicle background.Formulations 3, 3A, 4, 5, 5A, 10, 10A, 13, 13A, 13B, 14, 14A, and 14Bshowed no or minor improvement in flux in comparison to the control API,while formulation 3B showed significant improvement of permeability.

b. Flux Ratio Comparison

Flux ratios obtained for the different formulations of3,4,3-L1(1,2-HOPO) are summarized in TABLE 10.6 and charted in FIG. 22 .The only formulation 3B resulting in a 75-fold permeability increase wasobtained with 10 mg/mL Polysorbate 80 and 1 mg/mL API, with a recordedpH of 3.72.

TABLE 10.6 FLUX RATIO COMPARISONS FOR TESTED FORMULATIONS FormulationCode Flux Ratio SD F0 (API Control) 1.00 F0 Repeat 1.00 0.09 F3 2.220.96 F3 Repeat 0.77 0.08 F3A 0.94 0.09 F3B 75.57 5.22 F4 1.04 0.43 F51.06 0.72 F5 Repeat 1.86 0.22 F5A 1.39 0.14 F10 0.84 0.45 F10A 0.77 0.09F13 2.3 0.93 F13 Repeat 0.71 0.12 F13A 0.95 0.07 F13B 1.04 0.08 F14 1.180.6 F14 Repeat 1.15 0.12 F14A 0.97 0.3 F14B 1.63 0.436. Part C Conclusion

Fifteen different permeation enhancers were evaluated for their abilityat increasing the permeability of 3,4,3-L1(1,2-HOPO), using an in vitroPAMPA assay with artificial GIT lipid membranes. A significant increasewas observed in permeability for one formulation containing 10 mg/mL ofPolysorbate 80 and 1 mg/mL of API. A11 other tested formulations showedno or minor improvement in permeability. Formulations containingPolysorbate 80 will be evaluated further in in vivo studies.

Analysis of 3,4,3-L1(1,2-HOPO) Lot ML-11-276 was performed and acertificate of analysis was prepared regarding appearance,identification by IR and ¹H-NMR, related compounds by HPLC, HPLC purity,heavy metal content, residual solvent content, water content by KarlFischer, residue on ignition, and purity.

TABLE 10.7 DISTRIBUTION COEFFICIENT MEASUREMENT SUMMARY Buffering Agent1: 7.5 E−02 M HCl Standard A1 Standard B1 A_(st) 0.885 A_(st) 1.906ε_(aq) 17735 ε_(aq) 18193 Samples A11 A12 A13 B11 B12 B13 pH 1.18 1.181.18 1.20 1.20 1.20 A_(aq) 1.578 1.587 1.592 1.446 1.436 1.483 C_(aq)8.90E−05 8.95E−05 8.98E−05 7.95E−05 7.89E−05 8.15E−05 C_(or) 1.08E−051.03E−05 1.00E−05 2.53E−05 2.58E−05 2.33E−05 D_(ow) 0.122 0.115 0.1120.318 0.327 0.285 Log(D_(ow)) −0.915 −0.938 −0.952 −0.497 −0.485 −0.545−0.935 0.011 −0.509 0.018 Buffering Agent 2: 7.5 E−03 M HCl Standard A2Standard B2 A_(st) 0.998 A_(st) 1.88 ε_(aq) 19999 ε_(aq) 17945 SamplesA11 A12 A13 B11 B12 B13 pH 2.15 2.15 2.15 2.16 2.16 2.16 A_(aq) 1.8261.807 1.798 1.251 1.261 1.280 C_(aq) 9.13E−05 9.04E−05 8.99E−05 6.97E−057.03E−05 7.13E−05 C_(or) 8.50E−06 9.45E−06 9.90E−06 3.51E−05 3.45E−053.34E−05 D_(ow) 0.093 0.105 0.110 0.503 0.491 0.469 Log(D_(ow)) −1.031−0.980 −0.958 −0.299 −0.309 −0.329 −0.990 0.022 −0.312 0.009 BufferingAgent 3: 7.5 E−04 M HCl Standard A3 Standard B3 A_(st) 0.75 A_(st) 1.892ε_(aq) 15029 ε_(aq) 18059 Samples A31 A32 A33 B31 B32 B33 pH 3.21 3.213.21 3.13 3.13 3.13 A_(aq) 1.271 1.282 1.267 1.834 1.808 1.852 C_(aq)8.46E−05 8.53E−05 8.43E−05 1.02E−04 1.00E−04 1.03E−04 C_(or) 1.52E−051.45E−05 1.55E−05 3.21E−06 4.65E−06 2.21E−06 D_(ow) 0.180 0.170 0.1840.032 0.046 0.022 Log(D_(ow)) −0.744 −0.769 −0.735 −1.500 −1.333 −1.666−0.750 0.010 −1.499 0.096 Buffering Agent 4: 1 E−02 M Acetic AcidStandard A4 Standard B4 A_(st) 0.885 A_(st) 1.883 ε_(aq) 17735 ε_(aq)17973 Samples A41 A42 A43 B41 B42 B43 pH 4.19 4.19 4.19 4.00 4.00 4.00A_(aq) 1.645 1.669 1.657 1.804 1.802 1.864 C_(aq) 9.28E−05 9.41E−059.34E−05 1.00E−04 1.00E−04 1.04E−04 C_(or) 7.05E−06 5.70E−06 6.37E−064.40E−06 4.51E−06 1.06E−06 D_(ow) 0.076 0.061 0.068 0.044 0.045 0.010Log(D_(ow)) −1.119 −1.218 −1.166 −1.359 −1.347 −1.922 −1.168 0.029−1.566 0.213 Buffering Agent 5: 1 E−02 M Acetic Acid Standard A5Standard B5 A_(st) 0.753 A_(st) 1.505 ε_(aq) 15090 ε_(aq) 14365 SamplesA51 A52 A53 B51 B52 B53 pH 5.13 5.13 5.13 5.20 5.20 5.20 A_(aq) 1.4431.466 1.434 1.425 1.391 1.365 C_(aq) 9.56E−05 9.72E−05 9.50E−05 9.92E−059.68E−05 9.50E−05 C_(or) 4.18E−06 2.65E−06 4.77E−06 5.57E−06 7.94E−069.75E−06 D_(ow) 0.044 0.027 0.050 0.056 0.082 0.103 Log(D_(ow)) −1.360−1.564 −1.299 −1.251 −1.086 −0.989 −1.408 0.080 −1.109 0.076 BufferingAgent 6: 1 E−02 M MES Standard A6 Standard B6 A_(st) 0.719 A_(st) 1.356ε_(aq) 14408 ε_(aq) 12943 Samples A61 A62 A63 B61 B62 B63 pH 6.30 6.306.30 6.15 6.15 6.15 A_(aq) 1.351 1.335 1.329 1.356 1.301 1.326 C_(aq)9.38E−05 9.27E−05 9.22E−05 1.05E−04 1.01E−04 1.02E−04 C_(or) 6.04E−067.15E−06 7.57E−06 0.00E+00 4.25E−06 2.32E−06 D_(ow) 0.064 0.077 0.0820.000 0.042 0.023 Log(D_(ow)) −1.191 −1.113 −1.086 #NUM! −1.374 −1.645−1.130 0.032 −1.510 0.136 Buffering Agent 7: 1 E−02 M HEPES Standard A7Standard B7 A_(st) 0.669 A_(st) 1.418 ε_(aq) 13406 ε_(aq) 13535 SamplesA71 A72 A73 B71 B72 B73 pH 6.75 6.75 6.75 6.83 6.83 6.83 A_(aq) 1.2681.302 1.319 1.341 1.381 1.374 C_(aq) 8.68E−05 8.91E−05 9.03E−05 1.00E−041.03E−04 1.03E−04 C_(or) 1.30E−05 1.07E−05 9.51E−06 4.27E−06 1.27E−061.80E−06 D_(ow) 0.150 0.120 0.105 0.043 0.012 0.017 Log(D_(ow)) −0.824−0.921 −0.977 −1.372 −1.910 −1.758 −0.908 0.045 −1.680 0.160 BufferingAgent 8: 1 E−02 M HEPES Standard A8 Standard B8 A_(st) 0.667 A_(st)1.516 ε_(aq) 13366 ε_(aq) 14470 Samples A81 A82 A83 B81 B82 B83 pH 7.427.42 7.42 7.46 7.46 7.46 A_(aq) 1.290 1.288 1.317 1.329 1.325 1.264C_(aq) 8.83E−05 8.82E−05 9.02E−05 9.96E−05 9.93E−05 9.47E−05 C_(or)1.15E−05 1.16E−05 9.65E−06 5.17E−06 5.47E−06 1.00E−05 D_(ow) 0.130 0.1320.107 0.052 0.055 0.106 Log(D_(ow)) −0.885 −0.879 −0.970 −1.285 −1.259−0.975 −0.912 0.029 −1.173 0.099 Buffering Agent 9: 1 E−02 M HEPESStandard A9 Standard B9 A_(st) 0.622 A_(st) 1.481 ε_(aq) 12464 ε_(aq)14136 Samples A91 A92 A93 B91 B92 B93 pH 8.00 8.00 8.00 8.00 8.00 8.00A_(aq) 1.313 1.357 1.288 1.367 1.391 1.377 C_(aq) 8.99E−05 9.29E−058.82E−05 1.02E−04 1.04E−04 1.03E−04 C_(or) 9.93E−06 6.91E−06 1.16E−052.32E−06 5.25E−07 1.57E−06 D_(ow) 0.110 0.074 0.132 0.023 0.005 0.015Log(D_(ow)) −0.957 −1.128 −0.879 −1.644 −2.298 −1.817 −0.988 0.074−1.920 0.196

TABLE 10.8 PAMPA MEASUREMENT SUMMARY Screening Stage 1 Average SampleC_(ACC), μm SD C_(ACC) Flux Ratio SD Ratio Formulation 1 <0.1Formulation 2 <0.1 Formulation 3 0.41 0.06 2.22 0.96 Formulation 4 0.190.02 1.04 0.43 Formulation 5 0.19 0.02 1.06 0.72 Formulation 6 SeeDiscussion Section Formulation 7 See Discussion Section Formulation 8<0.1 Formulation 9 <0.1 Formulation 10 0.15 0.05 0.84 0.45 Formulation11 <0.1 Formulation 12 <0.1 Formulation 13 0.42 0.02 2.30 0.93Formulation 14 0.22 0.07 1.18 0.60 Formulation 15 <0.1 Formulation-0Control 0.18 0.07 1.00 Sample C_(acc), μm SD C_(acc) Ratio SD RatioFormulation 3 0.12 0.01 0.77 0.08 Formulation 14B 0.25 0.06 1.63 0.43Formulation 3A 0.14 0.01 0.94 0.09 Formulation 3B 11.45 0.38 75.57 5.22Formulation 13 0.11 0.02 0.71 0.12 Formulation 5 0.28 0.03 1.86 0.22Formulation 13A 0.14 0.01 0.95 0.07 Formulation 5A 0.21 0.02 1.39 0.14Formulation 13B 0.16 0.01 1.04 0.08 Formulation 14 0.17 0.02 1.15 0.12Formulation 10A 0.12 0.01 0.77 0.09 Formulation 14A 0.15 0.04 0.97 0.30Formulation-0 Control 0.15 0.01 1.00 0.09 Formulation 4 <0.1 Formulation4A <0.1 Formulation 10 <0.1 Sample—compound name based on providedinformation Average C_(ACC)—average value of API concentrationdetermined in the acceptor compartment after 18 hours of incubation FluxRatio—calculated based on a ratio between UV signals in the acceptorcompartment when corresponding donor compartment has formulated productand pure API respectively SD C_(ACC)—standard deviation of concentrationfrom a triplicate measurements SD Ratio—standard deviation of a ratiocalculated taking into account propagation of errors

Example 11—Permeability Enhancement of 3,4,3-L1(1,2-HOPO) Summary

The objective of the analytical study described in this report was toevaluate additional oral permeation enhancers for their ability atenhancing the permeability of the active pharmaceutical ingredient3,4,3-L1(1,2-HOPO). Evaluation was performed using an in vitro pK assaybased on the Double-Sink™ PAMPA technology developed by pION, Inc.

Thirty one different permeation enhancers were evaluated, in addition tothe original fifteen described in study 12-003-C, for their ability atincreasing the permeability of 3,4,3-L1(1,2-HOPO), using an in vitroPAMPA assay with artificial GIT lipid membranes. Significant increaseswere observed in permeability for two formulations containing2-Octyl-1-dodecanol and sodium oleate, respectively. A11 other testedformulations showed no or minor improvement in permeability.Formulations containing Polysorbate 80 were re-evaluated and previousenhancement results (as described in 12-003-C) could not be reproduced.

1. Purpose of Study

The purpose of this study was to provide data that can be used tosupport research efforts. It was not conducted in accordance with U.S.Food and Drug Administration (FDA) “Good Laboratory Practice forNonclinical Laboratory Studies” (GLP) regulations, as described in 21CFR Part 58. However, the study was planned, performed, recorded, andreported in accordance with standard practices to ensure data qualityand integrity.

2. Objective of Study

The objective of this study was to evaluate additional oral permeationenhancers for their ability at enhancing the permeability of the activepharmaceutical ingredient 3,4,3-L1(1,2-HOPO). Evaluation was performedusing an in vitro pK assay based on the Double-Sink™ PAMPA technologydeveloped by pION, Inc. This a follow-up study to LBNL No. 12-003-C, inwhich fifteen oral permeation enhancers were initially tested.

3. Experimental Design

The permeability enhancement study was performed in two additionalstages (stages 3 and 4, following the stages 1 and 2 described inEXAMPLE 10). In stage 3, 32 formulations were prepared and screened(including 31 new formulations and 1 repeat of the most successfulformulation from EXAMPLE 10). Stage 4 was performed to refine theconcentrations of the permeability enhancers that seemed to displaypermeability enhancement in the first three screenings and verifyreproducibility. The screening conditions for both stages 3 and 4 arelisted below in TABLE 11.1 and TABLE 11.2, respectively. The samplesolutions were stored throughout the study in 20 mL clear scintillationglass vials with polypropylene cap and pulp foil liner, wrapped inaluminum foil.

TABLE 11.1 PAMPA SCREENING STAGE 3 PAMPA Screening Stage 3 FormulationVehicle Permeation Enhancer Preparation Max. IIG Enhancer's API's limitor Target Target Vehicle Formulation Enhancer Literature Conc ConcVolume Code Class Selected report (mg/mL) (mg/mL) (mL) F0 Repeat3,4,3-LI(1,2- None NA NA 1 10 2&3 HOPO)API F16 Anionic Sodium decylsulfate  0.010% 0.20 1 10 Surfactant F17 Anionic Sodium octyl sulfate0.01% 0.20 1 10 Surfactant F18 Cationic Decyltrimethyl- 0.10% 1.00 1 10Surfactant ammoniumbromide F19 Noninonic Span-80 (Sorbitan monooleate)1.7 MG 2.50 1 10 Surfactant (Oral tablet) F20 Noninonic Triton X-100 N/A2.50 1 10 Surfactant F21 Bile Salts Sodium glycocholate hydrate 30 mg/mL1.00 1 10 F22 Fatty Acid Cholic acid N/A 2.50 1 10 F23 Fatty AcidHeptanoic acid N/A 2.50 1 10 F24 Fatty Ester Isopropyl Palmitate 0.1 to1% 2.50 1 10 F25 Fatty Ester Methyl laurate 0.1 to 1% 2.50 1 10 F26Fatty Amine Sodium oleate N/A 2.50 1 10 F27 Sodium Urea 0.018 MG 2.50 110 Salts of (Oral tablet, Fatty Acid coated) F28 Nitrogen1-Octyl-2-pyrrolidone 0.1 to 1% 2.50 1 10 containing Rings F29 Nitrogen1-Methylpiperazine 0.1 to 1% 2.50 1 10 containing Rings F30 Nitrogen1-Methyl-2-Pyrrolidinone 0.1 to 1% 2.50 1 10 containing Rings F31Nitrogen n-Caproic Acid 0.1 to 1% 2.50 1 10 containing Rings F32 OthersSodium Salicylate N/A 2.50 1 10 F33 Others (=)-Limonene N/A 2.50 1 10F34 Others L-Fenchone N/A 2.50 1 10 F35 Others Cineole N/A 2.50 1 10 F36Others Pinene oxide N/A 2.50 1 10 F37 Others 2-Octyl-1-dodecanol N/A2.50 1 10 F38 Natural Cumin seed oil N/A 2.50 1 10 F39 Lipid CaproylPGMC N/A 5.00 1 10 Excipient F40 Lipid Caproyl 90 (Propylene glycol N/A5.00 1 10 Excipient dicaprylate) F41 Lipid Lauroglycol FCC N/A 5.00 1 10Excipient F42 Lipid Lauroglycol 80 N/A 5.00 1 10 Excipient F43 LipidLabrafac PG N/A 5.00 1 10 Excipient F44 Lipid Transcutol N/A 5.00 1 10Excipient F45 Lipid Gelucire 50/13 N/A 5.00 1 10 Excipient F46 LipidLabrafil M1944 CS N/A 5.00 1 10 Excipient F3B Repeat 1 NoninonicPolysorbate 80 N/A 10.00 1 10 Surfactant

TABLE 11.2 PAMPA SCREENING STAGE 4 PAMPA Screening Stage 4 PermeationEnhancer Formulation Vehicle Preparation Max. IIG Enhancer's API's limitor Target Target Vehicle Formulation Enhancer Literature Conc ConcVolume Code Class Selected report (mg/mL) (mg/mL) (mL) F0 Repeat 43,4,3-LI(1,2- None NA NA 1 10 HOPO) API F0 Repeat 5 3,4,3-LI(1,2- NoneNA NA 1 10 HOPO) API F3B Repeat 2 Noninonic Polysorbate 80 N/A 10.00 110 Surfactant F3B Repeat 3 Noninonic Polysorbate 80 N/A 10.00 1 10Surfactant F3B Repeat 4 Noninonic Polysorbate 80 N/A 10.00 1 10Surfactant F26 Repeat 1 Fatty Amine Sodium oleate N/A 2.50 1 10 F26Repeat 2 Fatty Amine Sodium oleate N/A 2.50 1 104. Materials And Methods

a. Test and Control Articles

-   Test Article: 3,4,3-L1(1,2-HOPO)-   Manufacturer: Ash Stevens, Inc. (Detroit, Mich.)-   Lot Number: ML-11-276-   Physical Description: Pale yellow solid-   Storage Conditions: Refrigerated 2-8° C. protected from light.    Materials:

Purified Water HPLC Grade - Supplier: Ricca Sodium lauryl sulfate (SLS)Spectrum chemicals Caprolactam Spexcertiprep Polysorbate 80 (Tween 80)Spectrum chemicals Sodium decyl sulfate Sigma-aldrich Sodium octylsulfate Sigma-aldrich Decyltrimethylammonium bromide Sigma-aldrichSpan-80 (Sorbitan monooleate) Sigma-aldrich Triton X-100 Sigma-aldrichSodium glycocholate hydrate Sigma-aldrich Cholic acid Sigma-aldrichHeptanoic acid Sigma-aldrich Isopropyl Palmitate Sigma-aldrich Methyllaurate Sigma-aldrich Sodium oleate TCI Urea Sigma-aldrich1-Octyl-2-pyrrolidone Sigma-aldrich 1-Methylpiperazine Sigma-aldrich1-Methyl-2-Pyrrolidinone Sigma-aldrich n-Caproic acid TCI SodiumSalicylate Sigma-aldrich (±)-Limonene TCI L-Fenchone Sigma-aldrichCineole Sigma-aldrich Pinene oxide Sigma-aldrich 2-Octyl-1-dodecanolSigma-aldrich Cumin seed oil Sigma-aldrich Caproyl PGMC GattefosseCaproyl 90 (Propylene glycol Gattefosse dicaprylate) Lauroglycol FCCGattefosse Lauroglycol 90 Gattefosse Labrafac PG Gattefosse TranscutolGattefosse Gelucire 50/13 Gattefosse Labrafil M1944 CS Gattefosse GIT-0lipid pIOn, Inc. Acceptor Sink Buffer pIOn, Inc. Prisma ™ Buffer pIOn,Inc. DMSO HPLC Grade, Burdick and Jackson Stirring device Gut-Box ™,pION, Inc.

-   Test Solutions: Vehicles containing the permeation enhancers were    prepared by weighing the adequate quantity of enhancer and    dissolving it into 100 mL of purified water to reach the designated    concentration. Test solutions were then prepared by weighing 10 mg    of 3,4,3-L1(1,2-HOPO), dissolving it into 10 mL of each of the    vehicles to reach a concentration of 1 mg/mL (purified water was    used for the control solution). The final pH and clarity of each    solution were recorded.

b. Sample Characterization

-   Visual Observation: For each sample solution, visual observation    consisted in recording color and clarity.-   pH Record: The pH of each sample solution prepared for permeability    analysis was measured and recorded.

c. Permeability Assay

-   In vitro PK assay based The PAMPA Evolution96™ instrument was used    for the liquid-   on Double-Sink™ handling, UV data collection and results processing.    The-   PAMPA assay layout: system consisted of a 96-well Double-Sink PAMPA    Sandwich with preloaded stirrers. A PAMPA sandwich was formed such    that each composite well was divided into two chambers, separated by    a 125 μm microfilter disc (0.45 μM pores), coated with Pion GIT-0    phospholipids mixture. Formulations were suspended in Prisma™    buffer. GIT-0 lipid painted on a filter support created an    artificial membrane separating two chamber of the permeation system    while the free of drug Acceptor Sink Buffer (ASB, pH 7.4) was placed    in the receiving compartment. After introducing the formulations in    the donor compartments, the PAMPA sandwich was incubated for 15-30    min or up to 24 hours and only the UV spectra of the receiver were    collected. Calibrated for in vivo conditions, individual-well    stirring was provided by the Gut-Box™ (Pion Inc.).    -   The appearance rate of a compound in the receiving compartment        of the PAMPA Sandwich containing formulation in the donor        compartment was compared to the corresponding rate in a        formulation-free system. The ratio between these two rates was        reported as a Flux Ratio.-   Sample Preparation: The formulations were gently shaken before the    assay and the aliquots of the samples were transferred in the donor    compartment of the PAMPA Sandwich. In order to verify that the    lipid-coated membrane was stable in the presence of the formulations    and to take into account possible affect of the formulation vehicle    on the UV spectra, the corresponding formulation vehicles solutions    containing no API were transferred in the donor compartment. The    samples and the corresponding vehicles were assayed in triplicates.    5. Results

a. PAMPA Assay Results

Observations (formulation appearance and pH) and PAMPA permeationresults are summarized in TABLE 11.3 for screening stages 1 and 2 and inTABLE 11.4 for screening stages 3 and 4. Based on the data obtained fromthe permeation assay, the GIT lipid-covered membranes were stable in thepresence of all tested formulations and formulation vehicles, and noleakage was detected. The API 3,4,3-L1(1,2-HOPO) indicated very lowpermeability, comparable or even lower than the permeability level ofthe references compound Ranitidine.

TABLE 11.3 PAMPA SCREENING RESULTS STAGES 1 AND 2 Formulation VehicleObservations/Results Preparation & pH API's Concentration: 1 mg/mL, 10in 100 mL mL Vehicle Purified Water Flux Ratio Enhancer's (In vitroTarget pK Formulation Enhancer Conc Quantity PAMPA Code Selected (mg/mL)(mg) pH Appearance pH Assay) F0 (Control) None NA NA 7.21 Clear Solution3.73 1.00 F0 Repeat NA NA 7.20 Clear Solution 3.70  1.00 ± 0.09 F1Sodium lauryl 0.10 10 6.58 Clear Solution 3.74 N/A sulfate F2Caprolactam 2.50 250 6.68 Clear Solution 3.72 N/A F3 Polysorbate 80 2.50250 6.07 Clear Solution 3.74  2.22 ± 0.96 F3 Repeat 2.50 250 6.34 ClearSolution 3.70  0.77 ± 0.08 F3A 5.00 500 6.30 Clear Solution 3.83  0.94 ±0.09 F3B 10.00 1000 6.54 Clear Solution 3.72 75.57 ± 5.22 F4 Sodium 2.50250 7.63 White 6.85  1.04 ± 0.43 deoxycholate dispersion F4 Repeat 2.50250 7.47 White 7.08 N/A dispersion F4A 10.00 1000 7.82 Opaque 7.07 N/Adispersion F5 Isopropyl myristate 2.50 250 6.55 Clear Solution 3.77 1.06 ± 0.72 F5 Repeat 2.50 250 6.73 Clear Solution 3.79  1.86 ± 0.22F5A 10.00 1000 6.82 Clear Solution 3.72  1.39 ± 0.14 with oily drops F61-Phenylpiperazine 2.50 250 9.99 Clear Solution 9.12 N/A F7 Piperine2.50 250 6.93 Clear Solution 3.79 N/A F8 Menthone 2.50 250 6.76 ClearSolution 3.72 N/A F9 Labrafac Lipophile 5.00 500 5.73 Oily globules 3.75N/A WL F10 Gelucire 44/14 5.00 500 3.87 Clear Solution 3.62  0.84 ± 0.45F10 Repeat 5.00 500 4.17 Clear Solution 3.72 N/A F10A 20.00 2000 3.41Opaque 3.44  0.77 ± 0.09 dispersion F11 Labrafil M2130 CS 5.00 500 5.65White 3.73 N/A dispersion F12 Labrafil M2125 CS 5.00 500 6.16 White 3.79N/A Dispersion F13 Maisine 35-1 5.00 500 6.23 Opaque 3.74  2.30 ± 0.93dispersion F13 Repeat 5.00 500 6.37 Opaque 3.67  0.71 ± 0.12 dispersionF13A 10.00 1000 6.35 White 3.68  0.95 ± 0.07 Dispersion F13B 20.00 20006.4.1 Opaque 3.92  1.04 ± 0.08 dispersion F14 Peceol 5.00 500 5.22 ClearSolution 3.70  1.18 ± 0.60 F14 Repeat 5.00 500 6.03 Clear Solution 3.74 1.15 ± 0.12 F14A 10.00 1000 6.08 Clear Solution 3.7  0.97 ± 0.30 F14B20.00 2000 6.03 Clear Solution 3.83  1.63 ± 0.43 F15 Labrasol 5.00 50005.27 White 3.71 N/A Dispersion

TABLE 11.4 PAMPA SCREENING RESULTS STAGES 3 AND 4 PAMPA ScreeningResults Stage 3 Formulation Vehicle Observations/Results Preparation &pH in 100 mL API's Concentration: 1 mg/mL, 10 Purified Water mL VehicleEnhancer's Flux Ratio Target (In vitro pK Formulation Conc QuantityPAMPA Code Enhancer Selected (mg/mL) (mg) pH Appearance pH Assay) F0Repeat 2 None NA NA 6.76 Clear 3.73  1.00 ± 0.55 Solution F0 Repeat 3None NA NA 6.76 Clear 3.73  1.00 ± 0.61 Solution F1 Sodium laurylsulfate 0.10 10 6.58 Clear 3.74 N/A (SLS) Solution F2 Caprolactam 2.50250 6.68 Clear 3.72 N/A Solution F3B Repeat Polysorbate 80 10.00 10006.29 Clear 3.89 N/A 1 Solution F16 Sodium decyl sulfate 0.20 20 5.99Clear 3.74  2.79 ± 2.09 Solution F17 Sodium octyl sulfate 0.20 20 6.08Clear 3.86 N/A Solution F18 Decyltrimethylammoniu 1.00 100 7.21 Clear3.68 N/A monooleate Solution F19 Span-80 (Sorbitan 2.50 250 6.27 White3.78  1.23 ± 0.75 monooleate) Dispersion F20 Triton X-100 2.50 250 5.27Clear 3.83 N/A Solution F21 Sodium glycocholate 1.00 100 7.06 Clear 4.241.13 ± 0.54 hydrate Solution F22 Cholic acid 2.50 250 4.67 White 3.86N/A Dispersion F23 Heptanoic acid 2.50 250 3.41 Clear 3.45 N/A SolutionF24 Isopropyl Palmitate 2.50 250 6.37 Dispersion 3.81 N/A F25 Methyllaurate 2.50 250 5.95 Oily 3.84  1.40 ± 1.07 Dispersion F26 Sodiumoleate 2.50 250 10.61 White 8.81 17.35 ± 6.77 Dispersion F27 Urea 2.50250 9.17 Clear 3.91 N/A Solution F28 1-Octy1-2-pyrrolidone 2.50 250 7.78Clear 3.77 N/A Solution F29 1-Methylpiperazine 2.50 250 10.91 Clear 9.80N/A Solution F30 1-Methyl-2- 2.50 250 6.84 Clear 3.82  1.79 ± 0.91Pyrrolidinone Solution F31 n-Caproic Acid 2.50 250 3.35 Clear 3.37  1.64± 1.79 Solution F32 Sodium Salicylate 2.50 250 6.80 Clear 4.32 N/ASolution F33 (±)-Limonene 2.50 250 6.41 Clear 3.72  2.79 ± 1.36 SolutionF34 L-Fenchone 2.50 250 7.38 Clear 3.71  0.57 ± 0.49 Solution F35Cineole 2.50 250 7.37 Clear 3.75  1.71 ± 0.73 Solution F36 Pinene oxide2.50 250 5.91 Clear 3.74  2.64 ± 1.27 Solution F37 2-Octy1-1-dodecanol2.50 250 7.26 Clear 3.75  3.14 ± 1.36 Solution F38 Cumin seed oil 2.50250 4.47 Clear 3.75 N/A Solution F39 Caproyl PGMC 5.00 500 6.44 OilySolution 3.75 N/A F40 Caproyl 90 (Propylene 5.00 500 6.59 Oily Solution3.73 N/A glycol dicaprylate) F41 Lauroglycol FCC 5.00 500 6.03 OilySolution 3.86 N/A F42 Lauroglycol 90 5.00 500 6.35 Oily Solution 3.77N/A F43 Labrafac PG 5.00 500 6.63 Oily Solution 3.74 N/A F44 Transcutol5.00 500 6.64 Oily Solution 3.77 N/A F45 Gelucire 50/13 5.00 500 5.95Opaque 3.84 N/A Solution F46 Labrafil M1944 CS 5.00 500 6.29 White 3.91N/A Dispersion PAMPA Screening Results Stage 4 Formulation VehiclePreparation & pH in 100 mL Purified Water Observations/Results Ehancer'sAPI's Concentration: 1 mg/mL, 10 Target mL Vehicle Formulation EnhancerConc Quantity Flux Flux Code Selected (mg/mL) (mg) Appearance pH Ratio 1Ratio 2 F0 Repeat 4 None NA NA 7.21 Clear  1.22 ± 0.66   1.00 ± 0.40solution F0 Repeat 5 NA NA 7.20 Clear  1.00 ± 0.65   0.82 ± 0.44solution F3B Polysorbate 80 10.00 1000 6.54 Clear 34.16 ± 16.61 28.06 ±9.05 Repeat 2 solution F3B 10.00 1000 6.29 Clear  1.51 ± 0.89   1.24 ±0.57 Repeat 3 solution F3B Repeat 10.00 1000 6.29 Clear  0.98 ± 0.50  0.81 ± 0.29 4 solution F26 Repeat Sodium oleate 2.50 250 10.61 White 8.22 ± 3.82   6.75 ± 1.95 1 Dispersion F26 Repeat 2.50 250 10.61 White12.16 ± 6.82   9.99 ± 4.27 2 Dispersion

For Formulations 17, 18, 20, 22, 23, 24, 27, 28, 29, 40, 42, 43, 44, 45,and 3B, the UV/Visible signal in the acceptor compartment was below thedetection limit and the flux ratio could not be determined. Formulations32 and 38 showed very high penetration rates for their correspondingvehicles that completely saturated the UV-Visible signal in the acceptorcompartment, precluding the signal detection of the API under the strongvehicle background. The significant improvement of permeability seenpreviously for formulation 3B was not reproducible in the subsequentrepeats. Significant and reproducible improvement was noted forformulation 26, and significant improvement was also observed forformulation 37. Formulations 16, 19, 21, 25, 30, 31, 33, 34, 35, and 36showed no or minor improvement in flux in comparison to the control API.

b. Flux Ratio Comparison

Flux ratios obtained for the different newly tested formulations of3,4,3-L1(1,2-HOPO) are summarized in TABLE 11.5. The repeats forformulation 3B did not reproduce the initial 75-fold permeabilityincrease. However, Formulation 26 did result in reproducibleenhancement, obtained with 2.50 mg/mL sodium oleate and 1 mg/mL API,with a recorded pH of 8.81.

TABLE 11.5 FLUX RATIO COMPARISONS FOR TESTED FORMULATIONS FormulationCode Flux Ratio SD F0 (API Control) 1.00 F0 Repeat 1 1.00 0.09 F0 Repeat2 1.00 F0 Repeat 3 1.00 0.09 F0 Repeat 4 1.22 0.66 1.00 0.40 F0 Repeat 51.00 0.65 0.82 0.44 F3 2.22 0.96 F3 Repeat 0.77 0.08 F3A 0.94 0.09 F3B75.57 5.22 F3B Repeat 1 NA NA F3B Repeat 2 34.16 16.61 28.06 9.05 F3BRepeat 3 1.51 0.89 1.24 0.57 F3B Repeat 4 0.98 0.50 0.81 0.29 F4 1.040.43 F5 1.06 0.72 F5 Repeat 1.86 0.22 F5A 1.39 0.14 F10 0.84 0.45 F10A0.77 0.09 F13 2.30 0.93 F13 Repeat 0.71 0.12 F13A 0.95 0.07 F13B 1.040.08 F14 1.18 0.60 F14 Repeat 1.15 0.12 F14A 0.97 0.30 F14B 1.63 0.43F16 2.79 2.09 F19 1.23 0.75 F21 1.13 0.54 F25 1.40 1.07 F26 17.35 6.77F26 Repeat 1 8.22 3.82 6.75 1.95 F26 Repeat 2 12.16 6.82 9.99 4.27 F301.79 0.91 F31 1.64 1.79 F33 2.79 1.36 F34 0.57 0.49 F35 1.71 0.73 F362.64 1.27 F37 3.14 1.366. Conclusion

Thirty one additional permeation enhancers were evaluated for theirability at increasing the permeability of 3,4,3-L1(1,2-HOPO), using anin vitro PAMPA assay with artificial GIT lipid membranes. Thesignificant increase in permeability originally observed for oneformulation containing 10 mg/mL of Polysorbate 80 and 1 mg/mL of API wasnot reproducible. While most other tested formulations showed no orminor improvement in permeability, improvement was noted forformulations containing 2.50 mg/mL sodium oleate or 2-octyl-1-dodecanol.Formulations containing sodium oleate or 2-octyl-1-dodecanol will beevaluated further in vivo.

Analysis of 3,4,3-L1(1,2-HOPO) Lot ML-11-276 was performed and acertificate of analysis was prepared regarding appearance,identification by IR and ¹H-NMR, related compounds by HPLC, HPLC purity,heavy metal content, residual solvent content, water content by KarlFischer, residue on ignition, and purity.

TABLE 11.6 PAMPA MEASUREMENT SUMMARY Screening Stage 3 Sample C_(ACC),μm SD C_(ACC) Flux Ratio SD Ratio Set 1 Formulation 16 0.30 0.19 2.792.09 Formulation 17 <0.1 Formulation 18 <0.1 Formulation 19 0.13 0.061.23 0.75 Formulation 20 <0.1 Formulation 21 0.12 0.03 1.13 0.54Formulation 22 <0.1 Formulation 23 <0.1 Formulation 24 <0.1 Formulation25 0.15 0.10 1.40 1.07 Formulation 26 1.85 0.09 17.35 6.77 Formulation27 <0.1 Formulation 0- 0.11 0.04 1.00 0.55 Control Set 2 Formulation 28<0.1 Formulation 29 <0.1 Formulation 30 0.25 0.07 1.79 0.91 Formulation31 0.23 0.23 1.64 1.79 Formulation 32 und Formulation 33 0.39 0.09 2.791.36 Formulation 34 0.08 0.06 0.57 0.49 Formulation 35 0.24 0.00 1.710.73 Formulation 36 0.37 0.08 2.64 1.27 Formulation 37 0.44 0.03 3.141.36 Formulation 38 und Formulation 0- 0.14 0.06 1.00 0.61 Control Set 3Formulation 39 0.14 0.04 Formulation 40 <0.1 Formulation 41 0.12 0.04Formulation 42 <0.1 Formulation 43 <0.1 Formulation 44 <0.1 Formulation45 <0.1 Formulation 46 0.27 0.10 Formulation 3B <0.1 Formulation 0- <0.1Control C_(ACC), SD Ratio SD Ratio SD Sample μm C_(ACC) (1) Ratio (2)Ratio F3B_2444 5.04 0.79 34.16 16.61 28.06 9.05 F3B_2487 0.22 0.08 1.510.89 1.24 0.57 F3B_2509 0.14 0.03 0.98 0.50 0.81 0.29 F26_2487 1.21 0.088.22 3.82 6.75 1.95 F26_2509 1.79 0.58 12.16 6.82 9.99 4.27F0-Control_2487 0.18 0.05 1.22 0.66 1.00 0.40 F0-Control_2509 0.15 0.071.00 0.65 0.82 0.44 Sample compound name based on provided information,four last digits numbers refer to the project number associated with theoriginally submitted samples. C_(ACC), μM average value of APIconcentration determined in the acceptor compartment after 18 hours ofincubation Flux Ratio calculated based on a ratio between UV signals inthe acceptor comparmient when corresponding donor compartment hasformulated product and pure API respectively Flux Ratio (1) calculatedbased on the control submitted for the project 132509 Flux Ratio(2) calculated based on the control submitted for the project 132487 SDC_(ACC) standard deviation of concentration from a replicatemeasurements SD Ratio standard deviation of a ratio calculated takinginto account propagation of errors.

Example 12

The feasibility of developing oral formulations for 3,4,3-L1(1,2-HOPO)was evaluated.

Four oral dosage forms were investigated: (i) powder in bottle, (ii)dispersible/dissolvable granules, (iii) chewable tablets, and (iv)conventional immediate release tablets. Based on the studies performed,nine formulation prototypes that showed immediate drug release behaviorand required physical properties were identified and selected for APIverification, gastric fluid dissolution, and related substance testingfollowing defined liquid chromatography methods. Among these selectedcompositions, two are powder in bottle formulations, two are granuleformulations, three are chewable tablet formulations, and two areconventional tablet formulations. The respective compositions of theseprototype formulations are summarized and tabulated below. All assaysconfirmed that these prototypes are suitable for further development.

Conventional Dosage Form → Powder in Bottle Granules Chewable TabletsTablets Ingredients□ Formulation ID → A2 A11 G11 G12 C11 C13 C21 T50 T51Intra-Granular Materials (for granules and conventional tablets)3,4,3-LI(1,2-HOPO) 1.000 1.000 1.000 1.000 0.500 0.500 0.500 0.500 0.500Sodium Oleate 0.092 0.092 0.092 0.092 0.046 0.046 0.046 0.046 0.046Microcrystalline Cellulose and — 1.000 — — — — — — — CarboxymethylCellulose, NF (Avivel RC-591) Croscarmellose Sodium, — — 0.075 0.0750.075 — 0.075 0.084 0.092 NF (Ac-Di-Sol) Microcrystalline Cellulose andguar — — 1.833 — 1.854 — 0.927 — — gum, NF (Avicel CE-15) LactoseMonohydrate, NF — — — 1.533 — — — — — (Pharmatose 300M) LactoseMonohydrate, Povidone and — — — — — 1.929 — — — Crospovidone, NF(Ludipress) Mannitol, USP (Mannogem) — — — — — — 0.9227 — — Magensiumsterate, NF (HyQual) — — — — 0.025 0.025 0.025 — — MicrocrystallineCellulose, — — — — — — — 0.410 0.501 NF (Avicel PH 102) Colloidalsilicone dioxide, — — — — — — — 0.005 0.006 NF (Cab-O-Sil M5P) Purifiedwater, USP — — Q.S. Q.S. — — — — — Magensium sterate, NF (HyQual) — — —— — — — — — Extra-Granular Materials (for granules and conventionaltablet) Hypromellose, 50 cps — — — 0.300 — — — — — Magensium sterate, NF(HyQual) — — — — — — — 0.005 0.006 Unit weight (g) 1.092 2.0962 3.0003.000 2.500 2.500 2.500 1.050 1.151

It is to be understood that, while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications withinthe scope of the invention will be apparent to those skilled in the artto which the invention pertains.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

What is claimed is:
 1. A pharmaceutical composition comprising: a1,2-HOPO chelating agent in an amount from about 100 to about 1500 mg;and sodium oleate, wherein the 1,2-HOPO chelating agent is3,4,3-L1-1,2-HOPO.
 2. The pharmaceutical composition of claim 1, whereinsodium oleate is present at about 70 to about 130 mg.
 3. Thepharmaceutical composition of claim 1, wherein sodium oleate is presentat 8 to 12% of a total weight of the composition.
 4. The pharmaceuticalcomposition of claim 3, wherein sodium oleate is about 11% of a totalweight of the composition.
 5. The pharmaceutical composition of claim 3,wherein the 3,4,3-L1-1,2-HOPO chelating agent is present in an amountfrom 300 to 1500 mg.
 6. The pharmaceutical composition of claim 4,wherein the 3,4,3-L1-1,2-HOPO chelating agent is present in an amountfrom 400 to 1200 mg.
 7. The pharmaceutical composition of claim 4,wherein the 3,4,3-L1-1,2-HOPO chelating agent is present in an amountfrom 100 to 300 mg.
 8. The pharmaceutical composition of claim 4,wherein the 3,4,3-L1-1,2-HOPO chelating agent is present in an amount of600 mg.
 9. The pharmaceutical composition of claim 1, wherein the3,4,3-L1-1,2-HOPO chelating agent is present in an amount from 300 to1500 mg.
 10. The pharmaceutical composition of claim 1, wherein the3,4,3-L1-1,2-HOPO chelating agent is present in an amount from 400 to1200 mg.
 11. The pharmaceutical composition of claim 1, wherein the3,4,3-L1-1,2-HOPO chelating agent is present in an amount from 100 to300 mg.
 12. The pharmaceutical composition of claim 1, wherein the3,4,3-L1-1,2-HOPO chelating agent is present in an amount of 600 mg. 13.The pharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is packaged as a tablet.
 14. The pharmaceutical compositionof claim 1, wherein the pharmaceutical composition is within a capsule.15. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is within one or more granules.
 16. Thepharmaceutical composition of claim 7, wherein the pharmaceuticalcomposition is packaged as a tablet.
 17. The pharmaceutical compositionof claim 7, wherein the pharmaceutical composition is within a capsule.18. The pharmaceutical composition of claim 7, wherein thepharmaceutical composition is within one or more granules.